Systems and methods for coordinating manufacturing of cells for patient-specific immunotherapy

ABSTRACT

A method for coordinating the manufacturing of an expanded cell therapy product for a patient may include receiving a cell order request to expand the cell therapy product for the patient; generating a patient-specific identifier or cell order identifier associated with the cell order request; and initiating a process to expand the cell therapy product from at least some of a solid tumor obtained from the patient. If acceptance parameters for the expansion cell therapy product do not meet certain acceptance criteria at a second time point subsequent to a first time point in the expansion process, it is determined whether re-performing the expansion of the cell therapy product using the cell expansion technique is possible from the first time point based on the acceptance parameters at the second time point. If such re-performing the expansion is possible, patient treatment events that use the expanded cell therapy product are rescheduled.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. patent application Ser. No.17/357,393, filed on Jun. 24, 2021, U.S. patent application Ser. No.17/357,360, filed on Jun. 24, 2021, U.S. patent application Ser. No.17/238,092, filed on Apr. 22, 2021, U.S. Provisional Application No.63/013,942, filed on Apr. 22, 2020, U.S. Provisional Application No.63/155,711, filed Mar. 2, 2021, and U.S. Provisional Application No.63/159,806, filed Mar. 11, 2021, each of which is incorporated herein byreference in its entirety for all purposes.

BACKGROUND

Treatment of bulky, refractory cancers using adoptive transfer of tumorinfiltrating lymphocytes (TILs) represents a powerful approach totherapy for patients with poor prognoses. Gattinoni, et al., Nat. Rev.Immunol. 2006, 6, 383-393. A large number of TILs are required forsuccessful immunotherapy, and a robust and reliable process is neededfor commercialization. This has been a challenge to achieve because oftechnical, logistical, and regulatory issues with cell expansion.IL-2-based TIL expansion followed by a “rapid expansion process” (REP)has become a preferred method for TIL expansion because of its speed andefficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al.,J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008,26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al.,J. Immunother. 2003, 26, 332-42. REP can result in a 1,000-foldexpansion of TILs over a 14-day period, although it requires a largeexcess (e.g., 200-fold) of irradiated allogeneic peripheral bloodmononuclear cells (PBMCs, also known as mononuclear cells (MNCs)), oftenfrom multiple donors, as feeder cells, as well as anti-CD3 antibody(OKT3) and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26,332-42. TILs that have undergone an REP procedure have producedsuccessful adoptive cell therapy following host immunosuppression inpatients with melanoma.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 schematically depicts various stages in a treatment of a patientusing adoptive cell therapy using TILs including various steps formanufacturing allogenic TILs.

FIG. 2A shows the timeline for a GEN 3 process for TIL manufacturing.

FIG. 2B shows a comparison between a 2A process and an embodiment of aGEN 3 process for TIL manufacturing.

FIG. 2C shows a comparison between an embodiment of a GEN 3, anembodiment of a GEN 3.1 process, and an alternate embodiment of a GEN 3.1 process for TIL manufacturing.

FIG. 3A shows a block diagram for a system for coordinating themanufacturing of TILs for a patient.

FIG. 3B illustrates the object schema for components of system 300 thatare suitably modified or built upon commercially available softwareplatforms in addition to those standard within those platforms inaccordance with some embodiments.

FIGS. 3C-3E schematically illustrate the tracking on biological materialthrough the manufacturing process at a manufacturing facility inaccordance with some embodiments.

FIG. 3F schematically illustrates the process for maintaining COC andCOI through the journey of the cell therapy product from obtaining thesolid tumor through the manufacturing process to infusion into thepatient in accordance with some embodiments of the manufacturing process(e.g., a GEN 3 process).

FIG. 3G is a representative image of a label for a cell therapy productin accordance with some embodiments.

FIG. 3H is a table showing various types of labels generated during theprocess of manufacturing cell therapy product in accordance with someembodiments.

FIGS. 3I and 3J are representative images of a label for a finishedproduct in accordance with some embodiments.

FIGS. 3K-3P are representative screenshot images of tumor procurementforms in accordance with some embodiments.

FIGS. 4A and 4B show a flow chart for determination of a schedule forpatient treatment events based on success of the TIL manufacturingprocess.

FIG. 4C shows a flow chart for an alternate embodiment for determinationof a schedule for patient treatment events based on success of the TILmanufacturing process.

FIG. 5 shows a diagram of an embodiment of process 2A, a 22-day processfor TIL manufacturing.

FIG. 6 shows a comparison between the 1C process and an embodiment ofthe 2A process for TIL manufacturing.

FIG. 7 shows the 1C process timeline.

FIG. 8 shows a detailed schematic for an embodiment of the 2A process.

FIG. 9 shows an exemplary Process 2A chart providing an overview ofSteps A through F.

FIG. 10 shows a comparison table of Steps A through F from exemplaryembodiments of process 1C and process 2A.

FIG. 11 shows a detailed comparison of an embodiment of process 1C andan embodiment of process 2A.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

SEQ ID NO:2 is the amino acid sequence of the light chain of muromonab.

SEQ ID NO:3 is the amino acid sequence of a recombinant human IL-2protein.

SEQ ID NO:4 is the amino acid sequence of aldesleukin.

SEQ ID NO:5 is an IL-2 form.

SEQ ID NO:6 is the amino acid sequence of nemvaleukin alfa.

SEQ ID NO:7 is an IL-2 form.

SEQ ID NO:8 is a mucin domain polypeptide.

SEQ ID NO:9 is the amino acid sequence of a recombinant human IL-4protein.

SEQ ID NO:10 is the amino acid sequence of a recombinant human IL-7protein.

SEQ ID NO:11 is the amino acid sequence of a recombinant human IL-15protein.

SEQ ID NO:12 is the amino acid sequence of a recombinant human IL-21protein.

SEQ ID NO:13 is an IL-2 sequence.

SEQ ID NO:14 is an IL-2 mutein sequence.

SEQ ID NO:15 is an IL-2 mutein sequence.

SEQ ID NO:16 is the HCDR1_IL-2 for IgG.IL2R67A.H1.

SEQ ID NO:17 is the HCDR2 for IgG.IL2R67A.H1.

SEQ ID NO:18 is the HCDR3 for IgG.IL2R67A.H1.

SEQ ID NO:19 is the HCDR1_IL-2 kabat for IgG.IL2R67A.H1.

SEQ ID NO:20 is the HCDR2 kabat for IgG.IL2R67A.H1.

SEQ ID NO:21 is the HCDR3 kabat for IgG.IL2R67A.H1.

SEQ ID NO:22 is the HCDR1_IL-2 clothia for IgG.IL2R67A.H1.

SEQ ID NO:23 is the HCDR2 clothia for IgG.IL2R67A.H1.

SEQ ID NO:24 is the HCDR3 clothia for IgG.IL2R67A.H1.

SEQ ID NO:25 is the HCDR1_IL-2 IMGT for IgG.IL2R67A.H1.

SEQ ID NO:26 is the HCDR2 IMGT for IgG.IL2R67A.H1.

SEQ ID NO:27 is the HCDR3 IMGT for IgG.IL2R67A.H1.

SEQ ID NO:28 is the V_(H) chain for IgG.IL2R67A.H1.

SEQ ID NO:29 is the heavy chain for IgG.IL2R67A.H1.

SEQ ID NO:30 is the LCDR1 kabat for IgG.IL2R67A.H1.

SEQ ID NO:31 is the LCDR2 kabat for IgG.IL2R67A.H1.

SEQ ID NO:32 is the LCDR3 kabat for IgG.IL2R67A.H1.

SEQ ID NO:33 is the LCDR1 chothia for IgG.IL2R67A.H1.

SEQ ID NO:34 is the LCDR2 chothia for IgG.IL2R67A.H1.

SEQ ID NO:35 is the LCDR3 chothia for IgG.IL2R67A.H1.

SEQ ID NO:36 is a V_(L) chain.

SEQ ID NO:37 is a light chain.

SEQ ID NO:38 is a light chain.

SEQ ID NO:39 is a light chain.

SEQ ID NO:40 is the amino acid sequence of human 4-1BB.

SEQ ID NO:41 is the amino acid sequence of murine 4-1BB.

SEQ ID NO:42 is the heavy chain for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:43 is the light chain for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:44 is the heavy chain variable region (V_(H)) for the 4-1BBagonist monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO:45 is the light chain variable region (V_(L)) for the 4-1BBagonist monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO:46 is the heavy chain CDR1 for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:47 is the heavy chain CDR2 for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:48 is the heavy chain CDR3 for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:49 is the light chain CDR1 for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:50 is the light chain CDR2 for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:51 is the light chain CDR3 for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:52 is the heavy chain for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:53 is the light chain for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:54 is the heavy chain variable region (VH) for the 4-1BBagonist monoclonal antibody urelumab (BMS-663513).

SEQ ID NO:55 is the light chain variable region (VL) for the 4-1BBagonist monoclonal antibody urelumab (BMS-663513).

SEQ ID NO:56 is the heavy chain CDR1 for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:57 is the heavy chain CDR2 for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:58 is the heavy chain CDR3 for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:59 is the light chain CDR1 for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:60 is the light chain CDR2 for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:61 is the light chain CDR3 for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:62 is an Fc domain for a TNFRSF agonist fusion protein.

SEQ ID NO:63 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:64 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:65 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:66 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:67 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:68 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:69 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:70 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:71 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:72 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:73 is an Fc domain for a TNFRSF agonist fusion protein.

SEQ ID NO:74 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:75 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:76 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:77 is a 4-1BB ligand (4-1BBL) amino acid sequence.

SEQ ID NO:78 is a soluble portion of 4-1BBL polypeptide.

SEQ ID NO:79 is a heavy chain variable region (V_(H)) for the 4-1BBagonist antibody 4B4-1-1 version 1.

SEQ ID NO:80 is a light chain variable region (V_(L)) for the 4-1BBagonist antibody 4B4-1-1 version 1.

SEQ ID NO:81 is a heavy chain variable region (V_(H)) for the 4-1BBagonist antibody 4B4-1-1 version 2.

SEQ ID NO:82 is a light chain variable region (V_(L)) for the 4-1BBagonist antibody 4B4-1-1 version 2.

SEQ ID NO:83 is a heavy chain variable region (V_(H)) for the 4-1BBagonist antibody H39E3-2.

SEQ ID NO:84 is a light chain variable region (V_(L)) for the 4-1BBagonist antibody H39E3-2.

SEQ ID NO:85 is the amino acid sequence of human OX40.

SEQ ID NO:86 is the amino acid sequence of murine OX40.

SEQ ID NO:87 is the heavy chain for the OX40 agonist monoclonal antibodytavolixizumab (MEDI-0562).

SEQ ID NO:88 is the light chain for the OX40 agonist monoclonal antibodytavolixizumab (MEDI-0562).

SEQ ID NO:89 is the heavy chain variable region (V_(H)) for the OX40agonist monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO:90 is the light chain variable region (V_(L)) for the OX40agonist monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO:91 is the heavy chain CDR1 for the OX40 agonist monoclonalantibody tavolixizumab (MEDI-0562).

SEQ ID NO:92 is the heavy chain CDR2 for the OX40 agonist monoclonalantibody tavolixizumab (MEDI-0562).

SEQ ID NO:93 is the heavy chain CDR3 for the OX40 agonist monoclonalantibody tavolixizumab (MEDI-0562).

SEQ ID NO:94 is the light chain CDR1 for the OX40 agonist monoclonalantibody tavolixizumab (MEDI-0562).

SEQ ID NO:95 is the light chain CDR2 for the OX40 agonist monoclonalantibody tavolixizumab (MEDI-0562).

SEQ ID NO:96 is the light chain CDR3 for the OX40 agonist monoclonalantibody tavolixizumab (MEDI-0562).

SEQ ID NO:97 is the heavy chain for the OX40 agonist monoclonal antibody11D4.

SEQ ID NO:98 is the light chain for the OX40 agonist monoclonal antibody11D4.

SEQ ID NO:99 is the heavy chain variable region (V_(H)) for the OX40agonist monoclonal antibody 11D4.

SEQ ID NO:100 is the light chain variable region (V_(L)) for the OX40agonist monoclonal antibody 11D4.

SEQ ID NO:101 is the heavy chain CDR1 for the OX40 agonist monoclonalantibody 11D4.

SEQ ID NO:102 is the heavy chain CDR2 for the OX40 agonist monoclonalantibody 11D4.

SEQ ID NO:103 is the heavy chain CDR3 for the OX40 agonist monoclonalantibody 11D4.

SEQ ID NO:104 is the light chain CDR1 for the OX40 agonist monoclonalantibody 11D4.

SEQ ID NO:105 is the light chain CDR2 for the OX40 agonist monoclonalantibody 11D4.

SEQ ID NO:106 is the light chain CDR3 for the OX40 agonist monoclonalantibody 11D4.

SEQ ID NO:107 is the heavy chain for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:108 is the light chain for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:109 is the heavy chain variable region (V_(H)) for the OX40agonist monoclonal antibody 18D8.

SEQ ID NO:110 is the light chain variable region (V_(L)) for the OX40agonist monoclonal antibody 18D8.

SEQ ID NO:111 is the heavy chain CDR1 for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:112 is the heavy chain CDR2 for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:113 is the heavy chain CDR3 for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:114 is the light chain CDR1 for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:115 is the light chain CDR2 for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:116 is the light chain CDR3 for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:117 is the heavy chain variable region (V_(H)) for the OX40agonist monoclonal antibody Hu119-122.

SEQ ID NO:118 is the light chain variable region (V_(L)) for the OX40agonist monoclonal antibody Hu119-122.

SEQ ID NO:119 is the heavy chain CDR1 for the OX40 agonist monoclonalantibody Hu119-122.

SEQ ID NO:120 is the heavy chain CDR2 for the OX40 agonist monoclonalantibody Hu119-122.

SEQ ID NO:121 is the heavy chain CDR3 for the OX40 agonist monoclonalantibody Hu119-122.

SEQ ID NO:122 is the light chain CDR1 for the OX40 agonist monoclonalantibody Hu119-122.

SEQ ID NO:123 is the light chain CDR2 for the OX40 agonist monoclonalantibody Hu119-122.

SEQ ID NO:124 is the light chain CDR3 for the OX40 agonist monoclonalantibody Hu119-122.

SEQ ID NO:125 is the heavy chain variable region (V_(H)) for the OX40agonist monoclonal antibody Hu106-222.

SEQ ID NO:126 is the light chain variable region (V_(L)) for the OX40agonist monoclonal antibody Hu106-222.

SEQ ID NO:127 is the heavy chain CDR1 for the OX40 agonist monoclonalantibody Hu106-222.

SEQ ID NO:128 is the heavy chain CDR2 for the OX40 agonist monoclonalantibody Hu106-222.

SEQ ID NO:129 is the heavy chain CDR3 for the OX40 agonist monoclonalantibody Hu106-222.

SEQ ID NO:130 is the light chain CDR1 for the OX40 agonist monoclonalantibody Hu106-222.

SEQ ID NO:131 is the light chain CDR2 for the OX40 agonist monoclonalantibody Hu106-222.

SEQ ID NO:132 is the light chain CDR3 for the OX40 agonist monoclonalantibody Hu106-222.

SEQ ID NO:133 is an OX40 ligand (OX40L) amino acid sequence.

SEQ ID NO:134 is a soluble portion of OX40L polypeptide.

SEQ ID NO:135 is an alternative soluble portion of OX40L polypeptide.

SEQ ID NO:136 is the heavy chain variable region (V_(H)) for the OX40agonist monoclonal antibody 008.

SEQ ID NO:137 is the light chain variable region (V_(L)) for the OX40agonist monoclonal antibody 008.

SEQ ID NO:138 is the heavy chain variable region (V_(H)) for the OX40agonist monoclonal antibody 011.

SEQ ID NO:139 is the light chain variable region (V_(L)) for the OX40agonist monoclonal antibody 011.

SEQ ID NO:140 is the heavy chain variable region (V_(H)) for the OX40agonist monoclonal antibody 021.

SEQ ID NO:141 is the light chain variable region (V_(L)) for the OX40agonist monoclonal antibody 021.

SEQ ID NO:142 is the heavy chain variable region (V_(H)) for the OX40agonist monoclonal antibody 023.

SEQ ID NO:143 is the light chain variable region (V_(L)) for the OX40agonist monoclonal antibody 023.

SEQ ID NO:144 is the heavy chain variable region (V_(H)) for an OX40agonist monoclonal antibody.

SEQ ID NO:145 is the light chain variable region (V_(L)) for an OX40agonist monoclonal antibody.

SEQ ID NO:146 is the heavy chain variable region (V_(H)) for an OX40agonist monoclonal antibody.

SEQ ID NO:147 is the light chain variable region (V_(L)) for an OX40agonist monoclonal antibody.

SEQ ID NO:148 is the heavy chain variable region (V_(H)) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:149 is the heavy chain variable region (V_(H)) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:150 is the light chain variable region (V_(L)) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:151 is the light chain variable region (V_(L)) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:152 is the heavy chain variable region (V_(H)) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:153 is the heavy chain variable region (V_(H)) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:154 is the light chain variable region (V_(L)) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:155 is the light chain variable region (V_(L)) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:156 is the heavy chain variable region (V_(H)) for an OX40agonist monoclonal antibody.

SEQ ID NO:157 is the light chain variable region (V_(L)) for an OX40agonist monoclonal antibody.

SEQ ID NO:158 is the heavy chain amino acid sequence of the PD-1inhibitor nivolumab.

SEQ ID NO:159 is the light chain amino acid sequence of the PD-1inhibitor nivolumab.

SEQ ID NO:160 is the heavy chain variable region (V_(H)) amino acidsequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:161 is the light chain variable region (V_(L)) amino acidsequence of the PD-1 inhibitor nivolumab.

SEQ ID NO:162 is the heavy chain CDR1 amino acid sequence of the PD-1inhibitor nivolumab.

SEQ ID NO:163 is the heavy chain CDR2 amino acid sequence of the PD-1inhibitor nivolumab.

SEQ ID NO:164 is the heavy chain CDR3 amino acid sequence of the PD-1inhibitor nivolumab.

SEQ ID NO:165 is the light chain CDR1 amino acid sequence of the PD-1inhibitor nivolumab.

SEQ ID NO:166 is the light chain CDR2 amino acid sequence of the PD-1inhibitor nivolumab.

SEQ ID NO:167 is the light chain CDR3 amino acid sequence of the PD-1inhibitor nivolumab.

SEQ ID NO:168 is the heavy chain amino acid sequence of the PD-1inhibitor pembrolizumab.

SEQ ID NO:169 is the light chain amino acid sequence of the PD-1inhibitor pembrolizumab.

SEQ ID NO:170 is the heavy chain variable region (V_(H)) amino acidsequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:171 is the light chain variable region (V_(L)) amino acidsequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO:172 is the heavy chain CDR1 amino acid sequence of the PD-1inhibitor pembrolizumab.

SEQ ID NO:173 is the heavy chain CDR2 amino acid sequence of the PD-1inhibitor pembrolizumab.

SEQ ID NO:174 is the heavy chain CDR3 amino acid sequence of the PD-1inhibitor pembrolizumab.

SEQ ID NO:175 is the light chain CDR1 amino acid sequence of the PD-1inhibitor pembrolizumab.

SEQ ID NO:176 is the light chain CDR2 amino acid sequence of the PD-1inhibitor pembrolizumab.

SEQ ID NO:177 is the light chain CDR3 amino acid sequence of the PD-1inhibitor pembrolizumab.

SEQ ID NO:178 is the heavy chain amino acid sequence of the PD-L1inhibitor durvalumab.

SEQ ID NO:179 is the light chain amino acid sequence of the PD-L1inhibitor durvalumab.

SEQ ID NO:180 is the heavy chain variable region (V_(H)) amino acidsequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:181 is the light chain variable region (V_(L)) amino acidsequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:182 is the heavy chain CDR1 amino acid sequence of the PD-L1inhibitor durvalumab.

SEQ ID NO:183 is the heavy chain CDR2 amino acid sequence of the PD-L1inhibitor durvalumab.

SEQ ID NO:184 is the heavy chain CDR3 amino acid sequence of the PD-L1inhibitor durvalumab.

SEQ ID NO:185 is the light chain CDR1 amino acid sequence of the PD-L1inhibitor durvalumab.

SEQ ID NO:186 is the light chain CDR2 amino acid sequence of the PD-L1inhibitor durvalumab.

SEQ ID NO:187 is the light chain CDR3 amino acid sequence of the PD-L1inhibitor durvalumab.

SEQ ID NO:188 is the heavy chain amino acid sequence of the PD-L1inhibitor avelumab.

SEQ ID NO:189 is the light chain amino acid sequence of the PD-L1inhibitor avelumab.

SEQ ID NO:190 is the heavy chain variable region (V_(H)) amino acidsequence of the PD-L1 inhibitor avelumab.

SEQ ID NO:191 is the light chain variable region (V_(L)) amino acidsequence of the PD-L1 inhibitor avelumab.

SEQ ID NO:192 is the heavy chain CDR1 amino acid sequence of the PD-L1inhibitor avelumab.

SEQ ID NO:193 is the heavy chain CDR2 amino acid sequence of the PD-L1inhibitor avelumab.

SEQ ID NO:194 is the heavy chain CDR3 amino acid sequence of the PD-L1inhibitor avelumab.

SEQ ID NO:195 is the light chain CDR1 amino acid sequence of the PD-L1inhibitor avelumab.

SEQ ID NO:196 is the light chain CDR2 amino acid sequence of the PD-L1inhibitor avelumab.

SEQ ID NO:197 is the light chain CDR3 amino acid sequence of the PD-L1inhibitor avelumab.

SEQ ID NO:198 is the heavy chain amino acid sequence of the PD-L1inhibitor atezolizumab.

SEQ ID NO:199 is the light chain amino acid sequence of the PD-L1inhibitor atezolizumab.

SEQ ID NO:200 is the heavy chain variable region (V_(H)) amino acidsequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:201 is the light chain variable region (V_(L)) amino acidsequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:202 is the heavy chain CDR1 amino acid sequence of the PD-L1inhibitor atezolizumab.

SEQ ID NO:203 is the heavy chain CDR2 amino acid sequence of the PD-L1inhibitor atezolizumab.

SEQ ID NO:204 is the heavy chain CDR3 amino acid sequence of the PD-L1inhibitor atezolizumab.

SEQ ID NO:205 is the light chain CDR1 amino acid sequence of the PD-L1inhibitor atezolizumab.

SEQ ID NO:206 is the light chain CDR2 amino acid sequence of the PD-L1inhibitor atezolizumab.

SEQ ID NO:207 is the light chain CDR3 amino acid sequence of the PD-L1inhibitor atezolizumab.

SEQ ID NO:208 is the heavy chain amino acid sequence of the CTLA-4inhibitor ipilimumab.

SEQ ID NO:209 is the light chain amino acid sequence of the CTLA-4inhibitor ipilimumab.

SEQ ID NO:210 is the heavy chain variable region (V_(H)) amino acidsequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:211 is the light chain variable region (V_(L)) amino acidsequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO:212 is the heavy chain CDR1 amino acid sequence of the CTLA-4inhibitor ipilimumab.

SEQ ID NO:213 is the heavy chain CDR2 amino acid sequence of the CTLA-4inhibitor ipilimumab.

SEQ ID NO:214 is the heavy chain CDR3 amino acid sequence of the CTLA-4inhibitor ipilimumab.

SEQ ID NO:215 is the light chain CDR1 amino acid sequence of the CTLA-4inhibitor ipilimumab.

SEQ ID NO:216 is the light chain CDR2 amino acid sequence of the CTLA-4inhibitor ipilimumab.

SEQ ID NO:217 is the light chain CDR3 amino acid sequence of the CTLA-4inhibitor ipilimumab.

SEQ ID NO:218 is the heavy chain amino acid sequence of the CTLA-4inhibitor tremelimumab.

SEQ ID NO:219 is the light chain amino acid sequence of the CTLA-4inhibitor tremelimumab.

SEQ ID NO:220 is the heavy chain variable region (V_(H)) amino acidsequence of the CTLA-4 inhibitor tremelimumab.

SEQ ID NO:221 is the light chain variable region (V_(L)) amino acidsequence of the CTLA-4 inhibitor tremelimumab.

SEQ ID NO:222 is the heavy chain CDR1 amino acid sequence of the CTLA-4inhibitor tremelimumab.

SEQ ID NO:223 is the heavy chain CDR2 amino acid sequence of the CTLA-4inhibitor tremelimumab.

SEQ ID NO:224 is the heavy chain CDR3 amino acid sequence of the CTLA-4inhibitor tremelimumab.

SEQ ID NO:225 is the light chain CDR1 amino acid sequence of the CTLA-4inhibitor tremelimumab.

SEQ ID NO:226 is the light chain CDR2 amino acid sequence of the CTLA-4inhibitor tremelimumab.

SEQ ID NO:227 is the light chain CDR3 amino acid sequence of the CTLA-4inhibitor tremelimumab.

SEQ ID NO:228 is the heavy chain amino acid sequence of the CTLA-4inhibitor zalifrelimab.

SEQ ID NO:229 is the light chain amino acid sequence of the CTLA-4inhibitor zalifrelimab.

SEQ ID NO:230 is the heavy chain variable region (V_(H)) amino acidsequence of the CTLA-4 inhibitor zalifrelimab.

SEQ ID NO:231 is the light chain variable region (V_(L)) amino acidsequence of the CTLA-4 inhibitor zalifrelimab.

SEQ ID NO:232 is the heavy chain CDR1 amino acid sequence of the CTLA-4inhibitor zalifrelimab.

SEQ ID NO:233 is the heavy chain CDR2 amino acid sequence of the CTLA-4inhibitor zalifrelimab.

SEQ ID NO:234 is the heavy chain CDR3 amino acid sequence of the CTLA-4inhibitor zalifrelimab.

SEQ ID NO:235 is the light chain CDR1 amino acid sequence of the CTLA-4inhibitor zalifrelimab.

SEQ ID NO:236 is the light chain CDR2 amino acid sequence of the CTLA-4inhibitor zalifrelimab.

SEQ ID NO:237 is the light chain CDR3 amino acid sequence of the CTLA-4inhibitor zalifrelimab.

DETAILED DESCRIPTION I. Introduction

Adoptive cell therapy utilizing TILs cultured ex vivo by the RapidExpansion Protocol (REP) has produced successful adoptive cell therapyfollowing host immunosuppression in patients with melanoma. For example,it has been found that in some cases, lymphodepletion prior to adoptivetransfer of tumor-specific T lymphocytes plays a key role in enhancingtreatment efficacy by eliminating regulatory T-cells and competingelements of the immune system (“cytokine sinks”). In some cases, theefficacy of the ACT may be increased by pre-treating the patientreceiving the ACT with non-myeloablative chemotherapy prior to aninfusion of TILs, e.g., 28-25 days prior to TIL infusion. It has alsobeen found that an IL-2 treatment regimen following TIL infusion mayimprove the chances of success of the therapy. The timing and durationof these pre-infusion treatment and post-infusion treatment determinesthe ultimate efficacy of entire therapeutic regimen.

Thus, scheduling various treatment regimens depends on the timing andduration of TIL manufacturing process which itself is dependent onacceptance parameters for the final product. Current infusion acceptanceparameters rely on readouts of the composition of TILs (e.g., CD28, CD8,or CD4 positivity) and on fold expansion and viability of the expandedTIL product (also referred to herein as a REP product). The foldexpansion and viability of the REP product, in turn, depends on variousparameters measured during the expansion process. Such variation in theoutput and timing of an acceptable REP product for infusion poseschallenges in logistics of the transport of the tumor to themanufacturing facility, transfer of the REP product and the schedulingof various patient treatment events during the TIL manufacturingprocess.

Moreover, various parameters such as cell viability and cell countduring different stages of the TIL manufacturing process determine theduration of subsequent stages so that an acceptable viability, numericalfold and final cell count is obtained at the end of the TILmanufacturing process.

In addition, it is important to keep track of the biological materialfrom the time it is removed from the patient to the time it is infusedinto the patient, including throughout the TIL manufacturing process, toavoid manufacturing delays, mislabeling of material andmisidentification of patient, and thereby improving patient safety.

The present disclosure provides a framework for coordinatingmanufacturing process of an expanded cell therapy product for a patientand the various patient treatment events by dynamically schedulingvarious patient treatment events based on various measured parametersduring different stages of the manufacturing process.

For example, in an embodiment, a method for coordinating themanufacturing of expanded T-cells for a patient may include receiving,by a computing, a cell order request to expand T-cells for the patient;generating, by the computing device, a patient-specific identifier orcell-order identifier associated with the cell order request; andinitiating a process to expand T-cells. The process to expand T-cellsmay include performing, at a clinical facility, a procedure on thepatient to obtain T-cells from the patient and transferring the obtainedT-cells to a manufacturing facility. After receiving the obtainedT-cells at the manufacturing facility, patient treatment events aredynamically scheduled by a computing device. The dynamic scheduling isdependent on acceptance parameters in subsequently obtained expansionT-cells. Expansion of T-cells from at least some of the obtained T-cellsusing a cell expansion technique is initiated and acceptance parametersin the expansion T-cells are determined.

In another embodiment, a method for coordinating the manufacturing of anexpanded cell therapy product for a patient may include receiving, by acomputing device, a cell order request to expand the cell therapyproduct for the patient; generating, by the computing device, apatient-specific identifier including a cell order identifier associatedwith the cell order request; and initiating a process to expand the celltherapy product. The process to expand the cell therapy product mayinclude performing, at a clinical facility, a procedure on the patientto obtain a solid tumor from the patient and transferring the obtainedsolid tumor to a manufacturing facility. After receiving the solid tumorat the manufacturing facility, the patient treatment events aredynamically scheduled by the computing device. The dynamic scheduling isdependent on acceptance parameters for subsequently obtained expansioncell therapy product. Expansion of the cell therapy product from atleast some of the obtained solid tumor using a cell expansion techniqueis initiated and acceptance parameters for the expansion cell therapyproduct are determined at a first time point and at a second time pointsubsequent to the first time point. It is determined whether theacceptance parameters for the expansion cell therapy product meetcertain acceptance criteria at the first time point and at the secondtime point. If the acceptance parameters at the first time point meetthe acceptance criteria, the expansion of the cell therapy product iscontinued up to the second time point. If the acceptance parameters forthe expansion cell therapy product do not meet the acceptance criteriaat the second time point, it is determined whether re-performing theexpansion of the cell therapy product using the cell expansion techniqueis possible from the first time point based on the acceptance parametersat the second time point. If re-performing the expansion is determinedto be possible, the expansion of the cell therapy product from at leastsome of the cell therapy product obtained at the second time point usingthe cell expansion technique from the first time point. A time ofcompletion of the expansion of the cell therapy product following there-performing of the expansion of the cell therapy product from thefirst time point is estimated. The patient treatment events arerescheduled based on the estimated time of completion of the expansionof the cell therapy product and a timing of patient treatment eventsprior to or subsequent to an infusion of the expanded cell therapyproduct in the patient. The subsequent expansion of the cell therapyproduct is completed from the first time point.

The present disclosure additionally provides methods and systems foraccurately tracking the biological material from the time it extractedfrom the patient until the time it is infused back in the patient. Inparticular, the methods and systems disclosed herein facilitatemaintenance of a chain of identity and a chain of custody for thebiological material.

For example, in an embodiment, a method of tracking a patient'sbiological material may include receiving a cell order request formanufacturing biological material for the patient. Upon receiving thecell order request, a computing device generates a patient-specificidentifier associated with the cell order request. The patient-specificidentifier may include one or more of a patient identifier, a cellrequest order identifier, an order code and a cell order lot number. Thepatient's biological material is then tracked during shipping thebiological material from a clinical facility where the biologicalmaterial is extracted from the patient to a manufacturing facility, atthe manufacturing facility during the manufacturing process and duringshipping the manufactured biological material from the manufacturingfacility to a clinical facility where the manufactured biologicalmaterial is and back to the clinical facility where the manufacturedbiological material is infused into the patient using thepatient-specific identifier. The tracking may include recording, by thecomputing device, a tracking event at each step from the shipping andmanufacturing processes. The record of the tracking events comprises achain of custody of the patient's biological material.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entireties.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body. In vitro assays encompass cell-based assays in whichcells alive or dead are employed and may also encompass a cell-freeassay in which no intact cells are employed.

The term “ex vivo” refers to an event which involves treating orperforming a procedure on a cell, tissue and/or organ which has beenremoved from a subject's body. Aptly, the cell, tissue and/or organ maybe returned to the subject's body in a method of surgery or treatment.

The term “rapid expansion” means an increase in the number ofantigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-,or 9-fold) over a period of a week, more preferably at least about10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a periodof a week, or most preferably at least about 100-fold over a period of aweek. A number of rapid expansion protocols are outlined below.

By “tumor infiltrating lymphocytes” or “TILs” herein is meant apopulation of cells originally obtained as white blood cells that haveleft the bloodstream of a subject and migrated into a tumor. TILsinclude, but are not limited to, CD8⁺ cytotoxic T-cells (lymphocytes),Th1 and Th17 CD4⁺ T-cells, natural killer cells, dendritic cells and M1macrophages. TILs include both primary and secondary TILs. “PrimaryTILs” are those that are obtained from patient tissue samples asoutlined herein (sometimes referred to as “freshly obtained” or “freshlyisolated”), and “secondary TILs” are any TIL cell populations that havebeen expanded or proliferated as discussed herein, including, but notlimited to bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”).TIL cell populations can include genetically modified TILs.

By “population of cells” (including TILs) herein is meant a number ofcells that share common traits. In general, populations generally rangefrom 1×10⁶ to 1×10¹⁰ in number, with different TIL populationscomprising different numbers. For example, initial growth of primaryTILs in the presence of IL-2 results in a population of bulk TILs ofroughly 1×10⁸ cells. REP expansion is generally done to providepopulations of 1.5×10⁹ to 1.5×10¹⁰ cells for infusion. In someembodiments, REP expansion is done to provide populations of2.3×10¹⁰-13.7×10¹⁰.

By “cryopreserved TILs” herein is meant that TILs, either primary, bulk,or expanded (REP TILs), are treated and stored in the range of about−150° C. to −60° C. General methods for cryopreservation are alsodescribed elsewhere herein, including in the Examples. For clarity,“cryopreserved TILs” are distinguishable from frozen tissue sampleswhich may be used as a source of primary TILs.

By “thawed cryopreserved TILs” herein is meant a population of TILs thatwas previously cryopreserved and then treated to return to roomtemperature or higher, including but not limited to cell culturetemperatures or temperatures wherein TILs may be administered to apatient.

TILs can generally be defined either biochemically, using cell surfacemarkers, or functionally, by their ability to infiltrate tumors andeffect treatment. TILs can be generally categorized by expressing one ormore of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56,CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively,TILs can be functionally defined by their ability to infiltrate solidtumors upon reintroduction into a patient.

The term “cryopreservation media” or “cryopreservation medium” refers toany medium that can be used for cryopreservation of cells. Such mediacan include media comprising 7% to 10% DMSO. Exemplary media includeCryoStor CS10, Hyperthermasol, as well as combinations thereof. The term“CS10” refers to a cryopreservation medium which is obtained fromStemcell Technologies or from Biolife Solutions. The CS10 medium may bereferred to by the trade name “CryoStor® CS10”. The CS10 medium is aserum-free, animal component-free medium which comprises DMSO.

The term “central memory T-cell” refers to a subset of T-cells that inthe human are CD45R0+ and constitutively express CCR7 (CCR7^(hi)) andCD62L (CD62^(hi)). The surface phenotype of central memory T-cells alsoincludes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors forcentral memory T-cells include BCL-6, BCL-6B, MBD2, and BMI1. Centralmemory T-cells primarily secret IL-2 and CD40L as effector moleculesafter TCR triggering. Central memory T-cells are predominant in the CD4compartment in blood, and in the human are proportionally enriched inlymph nodes and tonsils.

The term “effector memory T-cell” refers to a subset of human ormammalian T-cells that, like central memory T-cells, are CD45R0+, buthave lost the constitutive expression of CCR7 (CCR7^(lo)) and areheterogeneous or low for CD62L expression (CD62L^(lo)). The surfacephenotype of central memory T-cells also includes TCR, CD3, CD127(IL-7R), and IL-15R. Transcription factors for central memory T-cellsinclude BLIMP1. Effector memory T-cells rapidly secret high levels ofinflammatory cytokines following antigenic stimulation, includinginterferon-γ, IL-4, and IL-5. Effector memory T-cells are predominant inthe CD8 compartment in blood, and in the human are proportionallyenriched in the lung, liver, and gut. CD8+ effector memory T-cells carrylarge amounts of perforin.

The term “closed system” refers to a system that is closed to theoutside environment. Any closed system appropriate for cell culturemethods can be employed with the methods of the present invention.Closed systems include, for example, but are not limited to closedG-containers. Once a tumor segment is added to the closed system, thesystem is not opened to the outside environment until the TILs are readyto be administered to the patient.

The terms “fragmenting,” “fragment,” and “fragmented,” as used herein todescribe processes for disrupting a tumor, includes mechanicalfragmentation methods such as crushing, slicing, dividing, andmorcellating tumor tissue as well as any other method for disrupting thephysical structure of tumor tissue.

The term “fine needle aspirate” or FNA refers to a type of biopsyprocedure that can be employed for sampling or diagnostic procedures,including tumor sampling, in which a sample is taken but the tumor isnot removed or resected. In fine needle aspiration, a hollow needle, forexample 25-18 gauge, is inserted into the tumor or into an areacontaining the tumor and fluid and cells (including tissue) are obtainedfor further analysis or expansion, as described herein. With an FNA, thecells are removed without preserving the histological architecture ofthe tissue cells. An FNA can comprise TILs. In some instances, a fineneedle aspiration biopsy is performed using an ultrasound-guided fineneedle aspiration biopsy needle. FNA needles are commercially availablefrom Becton Dickinson, Covidien, and the like.

The term “core biopsy” or “core needle biopsy” refers to a type ofbiopsy procedure that can be employed for sampling or diagnosticprocedures, including tumor sampling, in which a sample is taken but thetumor is not removed or resected. In a core biopsy, a hollow needle, forexample 16-11 gauge, is inserted into the tumor or into an areacontaining the tumor and fluid and cells (including tissue) are obtainedfor further analysis or expansion, as described herein. With a corebiopsy, the cells can be removed with some preservation of thehistological architecture of the tissue cells, given the larger needlesize as compared to a FNA. The core biopsy needle is generally of agauge size that is able to preserve at least some portion of thehistological architecture of the tumor. A core biopsy can comprise TILs.In some instances, a core needle biopsy is performed using a biopsyinstrument, a vacuum-assisted core-needle biopsy instrument, asteretactically guided core-needle biopsy instrument, anultrasound-guided core-needle biopsy instrument, an MM-guidedcore-needle biopsy instrument commercially available from Bard Medical,Becton Dickinson, and the like.

The terms “peripheral blood mononuclear cells” and “PBMCs” refers to aperipheral blood cell having a round nucleus, including lymphocytes(T-cells, B cells, NK cells) and monocytes. When used asantigen-presenting cells (PBMCs are a type of antigen-presenting cell),the peripheral blood mononuclear cells are irradiated allogeneicperipheral blood mononuclear cells.

The terms “peripheral blood lymphocytes” and “PBLs” refer to T-cellsexpanded from peripheral blood. In some embodiments, PBLs are separatedfrom whole blood or apheresis product from a donor. In some embodiments,PBLs are separated from whole blood or apheresis product from a donor bypositive or negative selection of a T-cell phenotype, such as the T-cellphenotype of CD3+ CD45+.

The term “anti-CD3 antibody” refers to an antibody or variant thereof,e.g., a monoclonal antibody and including human, humanized, chimeric ormurine antibodies which are directed against the CD3 receptor in theT-cell antigen receptor of mature T-cells. Anti-CD3 antibodies includeOKT-3, also known as muromonab. Anti-CD3 antibodies also include theUHCT1 clone, also known as T3 and CD3c. Other anti-CD3 antibodiesinclude, for example, otelixizumab, teplizumab, and visilizumab.

The term “OKT-3” (also referred to herein as “OKT3”) refers to amonoclonal antibody or biosimilar or variant thereof, including human,humanized, chimeric, or murine antibodies, directed against the CD3receptor in the T-cell antigen receptor of mature T-cells, and includescommercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure,Miltenyi Biotech, Inc., San Diego, Calif., USA) and muromonab orvariants, conservative amino acid substitutions, glycoforms, orbiosimilars thereof. The amino acid sequences of the heavy and lightchains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2).A hybridoma capable of producing OKT-3 is deposited with the AmericanType Culture Collection and assigned the ATCC accession number CRL 8001.A hybridoma capable of producing OKT-3 is also deposited with EuropeanCollection of Authenticated Cell Cultures (ECACC) and assigned CatalogueNo. 86022706.

TABLE 1 Amino acid sequences of muromonab. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 1QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY  60Muromonab heavyNQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120chain KTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL180 YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG240 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE360 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQ ID NO: 2QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH  60Muromonab lightFRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS 120chain SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL180 TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC 213

The term “IL-2” (also referred to herein as “IL2”) refers to the T cellgrowth factor known as interleukin-2 and includes all forms of IL-2including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-2 isdescribed, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek,Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which areincorporated by reference herein. The amino acid sequence of recombinanthuman IL-2 suitable for use in the invention is given in Table 2 (SEQ IDNO:3). For example, the term IL-2 encompasses human, recombinant formsof IL-2 such as aldesleukin (PROLEUKIN, available commercially frommultiple suppliers in 22 million IU per single use vials), as well asthe form of recombinant IL-2 commercially supplied by CellGenix, Inc.,Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd.,East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercialequivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125human IL-2) is a nonglycosylated human recombinant form of IL-2 with amolecular weight of approximately 15 kDa. The amino acid sequence ofaldesleukin suitable for use in the invention is given in Table 2 (SEQID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, asdescribed herein, including the pegylated IL2 prodrug bempegaldesleukin(NKTR-214, pegylated human recombinant IL-2 as in SEQ ID NO:4 in whichan average of 6 lysine residues are N⁶ substituted with[(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl),which is available from Nektar Therapeutics, South San Francisco,Calif., USA, or which may be prepared by methods known in the art, suchas the methods described in Example 19 of International PatentApplication Publication No. WO 2018/132496 A1 or the method described inExample 1 of U.S. Patent Application Publication No. US 2019/0275133 A1,the disclosures of which are incorporated by reference herein.Bempegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitablefor use in the invention are described in U.S. Patent ApplicationPublication No. US 2014/0328791 A1 and International Patent ApplicationPublication No. WO 2012/065086 A1, the disclosures of which areincorporated by reference herein. Alternative forms of conjugated IL-2suitable for use in the invention are described in U.S. Pat. Nos.4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of whichare incorporated by reference herein. Formulations of IL-2 suitable foruse in the invention are described in U.S. Pat. No. 6,706,289, thedisclosure of which is incorporated by reference herein.

In some embodiments, an IL-2 form suitable for use in the presentinvention is THOR-707, available from Synthorx, Inc. The preparation andproperties of THOR-707 and additional alternative forms of IL-2 suitablefor use in the invention are described in U.S. Patent ApplicationPublication Nos. US 2020/0181220 A1 and US 2020/0330601 A1, thedisclosures of which are incorporated by reference herein. In someembodiments, and IL-2 form suitable for use in the invention is aninterleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2polypeptide; and a conjugating moiety that binds to the isolated andpurified IL-2 polypeptide at an amino acid position selected from K35,T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72,and Y107, wherein the numbering of the amino acid residues correspondsto SEQ ID NO:5. In some embodiments, the amino acid position is selectedfrom T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72,and Y107. In some embodiments, the amino acid position is selected fromT37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. Insome embodiments, the amino acid position is selected from T37, T41,F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the aminoacid position is selected from R38 and K64. In some embodiments, theamino acid position is selected from E61, E62, and E68. In someembodiments, the amino acid position is at E62. In some embodiments, theamino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45,E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated tolysine, cysteine, or histidine. In some embodiments, the amino acidresidue is mutated to cysteine. In some embodiments, the amino acidresidue is mutated to lysine. In some embodiments, the amino acidresidue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62,E68, K64, P65, V69, L72, and Y107 is further mutated to an unnaturalamino acid. In some embodiments, the unnatural amino acid comprisesN6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK),BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine,allyloxycarbonyllysine, 2-amino-8-oxononanoic acid,2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine,p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine,m-acetylphenylalanine, 2-amino-8-oxononanoic acid,p-propargyloxyphenylalanine, p-propargyl-phenylalanine,3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine,isopropyl-L-phenylalanine, p-azido-L-phenylalanine,p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine,p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine,O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine,phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine,phosphonoserine, L-3-(2-naphthyl)alanine,2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoicacid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine. In someembodiments, the IL-2 conjugate has a decreased affinity to IL-2receptor α (IL-2Rα) subunit relative to a wild-type IL-2 polypeptide. Insome embodiments, the decreased affinity is about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% decrease inbinding affinity to IL-2Ra relative to a wild-type IL-2 polypeptide. Insome embodiments, the decreased affinity is about 1-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold, ormore relative to a wild-type IL-2 polypeptide. In some embodiments, theconjugating moiety impairs or blocks the binding of IL-2 with IL-2Rα. Insome embodiments, the conjugating moiety comprises a water-solublepolymer. In some embodiments, the additional conjugating moietycomprises a water-soluble polymer. In some embodiments, each of thewater-soluble polymers independently comprises polyethylene glycol(PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol andpropylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ),poly(N-acryloylmorpholine), or a combination thereof. In someembodiments, each of the water-soluble polymers independently comprisesPEG. In some embodiments, the PEG is a linear PEG or a branched PEG. Insome embodiments, each of the water-soluble polymers independentlycomprises a polysaccharide. In some embodiments, the polysaccharidecomprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose,heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES). Insome embodiments, each of the water-soluble polymers independentlycomprises a glycan. In some embodiments, each of the water-solublepolymers independently comprises polyamine. In some embodiments, theconjugating moiety comprises a protein. In some embodiments, theadditional conjugating moiety comprises a protein. In some embodiments,each of the proteins independently comprises an albumin, a transferrin,or a transthyretin. In some embodiments, each of the proteinsindependently comprises an Fc portion. In some embodiments, each of theproteins independently comprises an Fc portion of IgG. In someembodiments, the conjugating moiety comprises a polypeptide. In someembodiments, the additional conjugating moiety comprises a polypeptide.In some embodiments, each of the polypeptides independently comprises aXTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PASpolypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or agelatin-like protein (GLK) polymer. In some embodiments, the isolatedand purified IL-2 polypeptide is modified by glutamylation. In someembodiments, the conjugating moiety is directly bound to the isolatedand purified IL-2 polypeptide. In some embodiments, the conjugatingmoiety is indirectly bound to the isolated and purified IL-2 polypeptidethrough a linker. In some embodiments, the linker comprises ahomobifunctional linker. In some embodiments, the homobifunctionallinker comprises Lomant's reagent dithiobis (succinimidylpropionate)DSP, 3′3′-dithiobis(sulfosuccinimidyl proprionate) (DTSSP),disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS),disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST),ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate(DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA),dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-dithiobispropionimidate (DTBP),1,4-di-(3′-(2′-pyridyldithio)propionamido)butane (DPDPB),bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), suchas e.g. 1,5-difluoro-2,4-dinitrobenzene or1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenylsulfone(DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED),formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipicacid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine,benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid,N,N′-ethylene-bis(iodoacetamide), orN,N′-hexamethylene-bis(iodoacetamide). In some embodiments, the linkercomprises a heterobifunctional linker. In some embodiments, theheterobifunctional linker comprises N-succinimidyl3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chainN-succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP),succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT),sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate(sulfo-LC-sMPT),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC),sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs),m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs),N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB),sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB),succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB),sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB),N-(γ-maleimidobutyryloxy)succinimide ester (GMBs),N-(y-maleimidobutyryloxy) sulfosuccinimide ester (sulfo-GMBs),succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC),succinimidyl6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate(sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive andsulfhydryl-reactive cross-linkers such as 4-(4-N-maleimidophenyl)butyricacid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H),3-(2-pyridyldithio)propionyl hydrazide (PDPH),N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA),N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA),sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA),sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate(sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB),N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB),N-succinimidyl-6-(4′-azido-2′-nitrophenyl amino)hexanoate (sANPAH),sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate(sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs),sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate(sAND), N-succinimidyl-4(4-azidophenyl)1,3′-dithiopropionate (sADP),N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sulfo-sADP),sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB),sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate(sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate(sulfo-5AMCA), p-nitrophenyl diazopyruvate (pNPDP),p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP),1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB),N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio) propionamide(APDP), benzophenone-4-iodoacetamide, p-azidobenzoyl hydrazide (ABH),4-(ρ-azidosalicylamido)butylamine (AsBA), or p-azidophenyl glyoxal(APG). In some embodiments, the linker comprises a cleavable linker,optionally comprising a dipeptide linker. In some embodiments, thedipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. Insome embodiments, the linker comprises a non-cleavable linker. In someembodiments, the linker comprises a maleimide group, optionallycomprising maleimidocaproyl (mc),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), orsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC). In some embodiments, the linker further comprises aspacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol(PAB), p-aminobenzyoxycarbonyl (PABC), a derivative, or an analogthereof. In some embodiments, the conjugating moiety is capable ofextending the serum half-life of the IL-2 conjugate. In someembodiments, the additional conjugating moiety is capable of extendingthe serum half-life of the IL-2 conjugate. In some embodiments, the IL-2form suitable for use in the invention is a fragment of any of the IL-2forms described herein. In some embodiments, the IL-2 form suitable foruse in the invention is pegylated as disclosed in U.S. PatentApplication Publication No. US 2020/0181220 A1 and U.S. PatentApplication Publication No. US 2020/0330601 A1. In some embodiments, theIL-2 form suitable for use in the invention is an IL-2 conjugatecomprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine(AzK) covalently attached to a conjugating moiety comprising apolyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises anamino acid sequence having at least 80% sequence identity to SEQ IDNO:5; and the AzK substitutes for an amino acid at position K35, F42,F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to theamino acid positions within SEQ ID NO:5. In some embodiments, the IL-2polypeptide comprises an N-terminal deletion of one residue relative toSEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in theinvention lacks IL-2R alpha chain engagement but retains normal bindingto the intermediate affinity IL-2R beta-gamma signaling complex. In someembodiments, the IL-2 form suitable for use in the invention is an IL-2conjugate comprising: an IL-2 polypeptide comprising anN6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugatingmoiety comprising a polyethylene glycol (PEG), wherein: the IL-2polypeptide comprises an amino acid sequence having at least 90%sequence identity to SEQ ID NO:5; and the AzK substitutes for an aminoacid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69,or L72 in reference to the amino acid positions within SEQ ID NO:5. Insome embodiments, the IL-2 form suitable for use in the invention is anIL-2 conjugate comprising: an IL-2 polypeptide comprising anN6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugatingmoiety comprising a polyethylene glycol (PEG), wherein: the IL-2polypeptide comprises an amino acid sequence having at least 95%sequence identity to SEQ ID NO:5; and the AzK substitutes for an aminoacid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69,or L72 in reference to the amino acid positions within SEQ ID NO:5. Insome embodiments, the IL-2 form suitable for use in the invention is anIL-2 conjugate comprising: an IL-2 polypeptide comprising anN6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugatingmoiety comprising a polyethylene glycol (PEG), wherein: the IL-2polypeptide comprises an amino acid sequence having at least 98%sequence identity to SEQ ID NO:5; and the AzK substitutes for an aminoacid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69,or L72 in reference to the amino acid positions within SEQ ID NO:5.

In some embodiments, an IL-2 form suitable for use in the invention isnemvaleukin alfa, also known as ALKS-4230 (SEQ ID NO:6), which isavailable from Alkermes, Inc. Nemvaleukin alfa is also known as humaninterleukin 2 fragment (1-59), variant (Cys¹²⁵>Ser⁵¹), fused viapeptidyl linker (⁶⁰GG⁶¹) to human interleukin 2 fragment (62-132), fusedvia peptidyl linker (¹³³GSGGGS¹³⁸) to human interleukin 2 receptora-chain fragment (139-303), produced in Chinese hamster ovary (CHO)cells, glycosylated; human interleukin 2 (IL-2) (75-133)-peptide[Cys¹²⁵(51)>Ser]-mutant (1-59), fused via a G₂ peptide linker (60-61) tohuman interleukin 2 (IL-2) (4-74)-peptide (62-132) and via a GSG₃Speptide linker (133-138) to human interleukin 2 receptor α-chain (IL2Rsubunit alpha, IL2Rα, IL2RA) (1-165)-peptide (139-303), produced inChinese hamster ovary (CHO) cells, glycoform alfa. The amino acidsequence of nemvaleukin alfa is given in SEQ ID NO:6. In someembodiments, nemvaleukin alfa exhibits the following post-translationalmodifications: disulfide bridges at positions: 31-116, 141-285, 184-242,269-301, 166-197 or 166-199, 168-199 or 168-197 (using the numbering inSEQ ID NO:6), and glycosylation sites at positions: N187, N206, T212using the numbering in SEQ ID NO:6. The preparation and properties ofnemvaleukin alfa, as well as additional alternative forms of IL-2suitable for use in the invention, is described in U.S. PatentApplication Publication No. US 2021/0038684 A1 and U.S. Pat. No.10,183,979, the disclosures of which are incorporated by referenceherein. In some embodiments, an IL-2 form suitable for use in theinvention is a protein having at least 80%, at least 90%, at least 95%,or at least 90% sequence identity to SEQ ID NO:6. In some embodiments,an IL-2 form suitable for use in the invention has the amino acidsequence given in SEQ ID NO:6 or conservative amino acid substitutionsthereof. In some embodiments, an IL-2 form suitable for use in theinvention is a fusion protein comprising amino acids 24-452 of SEQ IDNO:7, or variants, fragments, or derivatives thereof. In someembodiments, an IL-2 form suitable for use in the invention is a fusionprotein comprising an amino acid sequence having at least 80%, at least90%, at least 95%, or at least 90% sequence identity to amino acids24-452 of SEQ ID NO:7, or variants, fragments, or derivatives thereof.Other IL-2 forms suitable for use in the present invention are describedin U.S. Pat. No. 10,183,979, the disclosures of which are incorporatedby reference herein. Optionally, in some embodiments, an IL-2 formsuitable for use in the invention is a fusion protein comprising a firstfusion partner that is linked to a second fusion partner by a mucindomain polypeptide linker, wherein the first fusion partner is IL-1Ra ora protein having at least 98% amino acid sequence identity to IL-1Ra andhaving the receptor antagonist activity of IL-Ra, and wherein the secondfusion partner comprises all or a portion of an immunoglobulincomprising an Fc region, wherein the mucin domain polypeptide linkercomprises SEQ ID NO:8 or an amino acid sequence having at least 90%sequence identity to SEQ ID NO:8 and wherein the half-life of the fusionprotein is improved as compared to a fusion of the first fusion partnerto the second fusion partner in the absence of the mucin domainpolypeptide linker.

TABLE 2 Amino acid sequences of interleukins. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 3MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL  60recombinantEEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120human IL-2 RWITFCQSII STLT 134 (rhIL-2) SEQ ID NO: 4PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE  60AldesleukinELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120ITFSQSIIST LT 132 SEQ ID NO: 5APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE  60IL-2 formEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 120WITFCQSIIS TLT 133 SEQ ID NO: 6SKNFHLRPRD LISNINVIVL ELKGSETTFM CEYADETATI VEFLNRWITF SQSIISTLTG  60NemvaleukinGSSSTKKTQL QLEHLLLDLQ MILNGINNYK NPKLTRMLTF KFYMPKKATE LKHLQCLEEE 120alfa LKPLEEVLNL AQGSGGGSEL CDDDPPEIPH ATFKAMAYKE GTMLNCECKR GFRRIKSGSL180 YMLCTGNSSH SSWDNQCQCT SSATRNTTKQ VTPQPEEQKE RKTTEMQSPM QPVDQASLPG240 HCREPPPWEN EATERIYHFV VGQMVYYQCV QGYRALHRGP AESVCKMTHG KTRWTQPQLI300 CTG 303 SEQ ID NO: 7MDAMKRGLCC VLLLCGAVFV SARRPSGRKS SKMQAFRIWD VNQKTFYLRN NQLVAGYLQG  60IL-2 formPNVNLEEKID VVPIEPHALF LGIHGGKMCL SCVKSGDETR LQLEAVNITD LSENRKQDKR 120FAFIRSDSGP TTSFESAACP GWFLCTAMEA DQPVSLTNMP DEGVMVTKFY FQEDESGSGG 180ASSESSASSD GPHPVITESR ASSESSASSD GPHPVITESR EPKSSDKTHT CPPCPAPELL 240GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ 300YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 360EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS 420RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK 452 SEQ ID NO: 8 SESSASSDGP HPVITP 16 mucin domain polypeptide SEQ ID NO: 9MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASKNT TEKETFCRAA TVLRQFYSHH  60recombinantEKDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSC PVKEANQSTL ENFLERLKTI 120human IL-4 MREKYSKCSS 130 (rhIL-4) SEQ ID NO: 10MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA NKEGMFLFRA  60recombinantARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP TKSLEENKSL 120human IL-7 KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH 153 (rhIL-7)SEQ ID NO: 11MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV ISLESGDASI  60recombinant HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS115 human IL-15 (rhIL-15) SEQ ID NO: 12MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG  60recombinantNNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ 120human IL-21 HLSSRTHGSE DS 132 (rhIL-21)

In some embodiments, an IL-2 form suitable for use in the inventionincludes a antibody cytokine engrafted protein comprises a heavy chainvariable region (V_(H)), comprising complementarity determining regionsHCDR1, HCDR2, HCDR3; a light chain variable region (V_(L)), comprisingLCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereofengrafted into a CDR of the V_(H) or the V_(L), wherein the antibodycytokine engrafted protein preferentially expands T effector cells overregulatory T cells. In an embodiment, the antibody cytokine engraftedprotein comprises a heavy chain variable region (V_(H)), comprisingcomplementarity determining regions HCDR1, HCDR2, HCDR3; a light chainvariable region (V_(L)), comprising LCDR1, LCDR2, LCDR3; and an IL-2molecule or a fragment thereof engrafted into a CDR of the V_(H) or theV_(L), wherein the IL-2 molecule is a mutein, and wherein the antibodycytokine engrafted protein preferentially expands T effector cells overregulatory T cells. In an embodiment, the IL-2 regimen comprisesadministration of an antibody described in U.S. Patent ApplicationPublication No. US 2020/0270334 A1, the disclosures of which areincorporated by reference herein. In an embodiment, the antibodycytokine engrafted protein comprises a heavy chain variable region(V_(H)), comprising complementarity determining regions HCDR1, HCDR2,HCDR3; a light chain variable region (V_(L)), comprising LCDR1, LCDR2,LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDRof the V_(H) or the V_(L), wherein the IL-2 molecule is a mutein,wherein the antibody cytokine engrafted protein preferentially expands Teffector cells over regulatory T cells, and wherein the antibody furthercomprises an IgG class heavy chain and an IgG class light chain selectedfrom the group consisting of: a IgG class light chain comprising SEQ IDNO:39 and a IgG class heavy chain comprising SEQ ID NO:38; a IgG classlight chain comprising SEQ ID NO:37 and a IgG class heavy chaincomprising SEQ ID NO:29; a IgG class light chain comprising SEQ ID NO:39and a IgG class heavy chain comprising SEQ ID NO:29; and a IgG classlight chain comprising SEQ ID NO:37 and a IgG class heavy chaincomprising SEQ ID NO:38.

In an embodiment, an IL-2 molecule or a fragment thereof is engraftedinto HCDR1 of the V_(H), wherein the IL-2 molecule is a mutein. In anembodiment, an IL-2 molecule or a fragment thereof is engrafted intoHCDR2 of the V_(H), wherein the IL-2 molecule is a mutein. In anembodiment, an IL-2 molecule or a fragment thereof is engrafted intoHCDR3 of the V_(H), wherein the IL-2 molecule is a mutein. In anembodiment, an IL-2 molecule or a fragment thereof is engrafted intoLCDR1 of the V_(L), wherein the IL-2 molecule is a mutein. In anembodiment, an IL-2 molecule or a fragment thereof is engrafted intoLCDR2 of the V_(L), wherein the IL-2 molecule is a mutein. In anembodiment, an IL-2 molecule or a fragment thereof is engrafted intoLCDR3 of the V_(L), wherein the IL-2 molecule is a mutein.

The insertion of the IL-2 molecule can be at or near the N-terminalregion of the CDR, in the middle region of the CDR or at or near theC-terminal region of the CDR. In some embodiments, the antibody cytokineengrafted protein comprises an IL-2 molecule incorporated into a CDR,wherein the IL2 sequence does not frameshift the CDR sequence. In someembodiments, the antibody cytokine engrafted protein comprises an IL-2molecule incorporated into a CDR, wherein the IL-2 sequence replaces allor part of a CDR sequence. The replacement by the IL-2 molecule can bethe N-terminal region of the CDR, in the middle region of the CDR or ator near the C-terminal region the CDR. A replacement by the IL-2molecule can be as few as one or two amino acids of a CDR sequence, orthe entire CDR sequences.

In some embodiments, an IL-2 molecule is engrafted directly into a CDRwithout a peptide linker, with no additional amino acids between the CDRsequence and the IL-2 sequence. In some embodiments, an IL-2 molecule isengrafted indirectly into a CDR with a peptide linker, with one or moreadditional amino acids between the CDR sequence and the IL-2 sequence.

In some embodiments, the IL-2 molecule described herein is an IL-2mutein. In some instances, the IL-2 mutein comprising an R67Asubstitution. In some embodiments, the IL-2 mutein comprises the aminoacid sequence SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, theIL-2 mutein comprises an amino acid sequence in Table 1 in U.S. PatentApplication Publication No. US 2020/0270334 A1, the disclosure of whichis incorporated by reference herein.

In an embodiment, the antibody cytokine engrafted protein comprises anHCDR1 selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19,SEQ ID NO:22 and SEQ ID NO:25. In an embodiment, the antibody cytokineengrafted protein comprises an HCDR1 selected from the group consistingof SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13 and SEQ ID NO:16. In anembodiment, the antibody cytokine engrafted protein comprises an HCDR1selected from the group consisting of HCDR2 selected from the groupconsisting of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ IDNO:26. In an embodiment, the antibody cytokine engrafted proteincomprises an HCDR3 selected from the group consisting of SEQ ID NO:18,SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:27. In an embodiment, theantibody cytokine engrafted protein comprises a V_(H) region comprisingthe amino acid sequence of SEQ ID NO:28. In an embodiment, the antibodycytokine engrafted protein comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO:29. In an embodiment, the antibody cytokineengrafted protein comprises a V_(L) region comprising the amino acidsequence of SEQ ID NO:36. In an embodiment, the antibody cytokineengrafted protein comprises a light chain comprising the amino acidsequence of SEQ ID NO:37. In an embodiment, the antibody cytokineengrafted protein comprises a V_(H) region comprising the amino acidsequence of SEQ ID NO:28 and a V_(L) region comprising the amino acidsequence of SEQ ID NO:36. In an embodiment, the antibody cytokineengrafted protein comprises a heavy chain region comprising the aminoacid sequence of SEQ ID NO:29 and a light chain region comprising theamino acid sequence of SEQ ID NO:37. In an embodiment, the antibodycytokine engrafted protein comprises a heavy chain region comprising theamino acid sequence of SEQ ID NO:29 and a light chain region comprisingthe amino acid sequence of SEQ ID NO:39. In an embodiment, the antibodycytokine engrafted protein comprises a heavy chain region comprising theamino acid sequence of SEQ ID NO:38 and a light chain region comprisingthe amino acid sequence of SEQ ID NO:37. In an embodiment, the antibodycytokine engrafted protein comprises a heavy chain region comprising theamino acid sequence of SEQ ID NO:38 and a light chain region comprisingthe amino acid sequence of SEQ ID NO:39. In an embodiment, the antibodycytokine engrafted protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 ofU.S. Patent Application Publication No. 2020/0270334 A1, or variants,derivatives, or fragments thereof, or conservative amino acidsubstitutions thereof, or proteins with at least 80%, at least 90%, atleast 95%, or at least 98% sequence identity thereto. In an embodiment,the antibody components of the antibody cytokine engrafted proteindescribed herein comprise immunoglobulin sequences, framework sequences,or CDR sequences of palivizumab. In some embodiments, the antibodycytokine engrafted protein described herein has a longer serum half-lifethat a wild-type IL-2 molecule such as, but not limited to, aldesleukinor a comparable molecule. In an embodiment, the antibody cytokineengrafted protein described herein has a sequence as set forth in Table3.

TABLE 3Sequences of exemplary palivizumab antibody-IL-2 engrafted proteins.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 13MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML  60IL-2 TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE120 TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT 153 SEQ ID NO: 14APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTAML TFKFYMPKKA TELKHLQCLE  60IL-2 muteinEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 120WITFCQSIIS TLT 133 SEQ ID NO: 15APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLE  60IL-2 muteinEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 120WITFCQSIIS TLT 133 SEQ ID NO: 16GFSLAPTSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTEKEYM PKKATELKHL  60HCDR1_IL-2QCLEEELKPL EEVLNLAQSK NFHLRPRDLI SNINVIVLEL KGSETTFMCE YADETATIVE 120FLNRWITFCQ SIISTLTSTS GMSVG 145 SEQ ID NO: 17 DIWWDDKKDY NPSLKS  16HCDR2 SEQ ID NO: 18 SMITNWYFDV  10 HCDR3 SEQ ID NO: 19APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTAML TFKFYMPKKA TELKHLQCLE  60HCDRE_IL-2 kabatEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 120WITFCQSIIS TLTSTSGMSV G 141 SEQ ID NO: 20 DIWWDDKKDY NPSLKS  16HCDR2 kabat SEQ ID NO: 21 SMITNWYFDV  10  HCDR3 kabat SEQ ID NO: 22GFSLAPTSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTEKEYM PKKATELKHL  60HCDR1_IL-2QCLEEELKPL EEVLNLAQSK NFHLRPRDLI SNINVIVLEL KGSETTFMCE YADETATIVE 120clothia FLNRWITFCQ SIISTLTSTS GM 142 SEQ ID NO:23 WWDDK   5HCDR2 clothia SEQ ID NO: 24 SMITNWYFDV  10 HCDR3 clothia SEQ ID NO: 25GFSLAPTSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTEKEYM PKKATELKHL  60HCDR1_IL-2 IMGTQCLEEELKPL EEVLNLAQSK NFHLRPRDLI SNINVIVLEL KGSETTFMCE YADETATIVE 120FLNRWITFCQ SIISTLTSTS GMS 143 SEQ ID NO: 26 IWWDDKK   7 HCDR2 IMGTSEQ ID NO: 27 ARSMITNWYF DV  12 HCDR3 IMGT SEQ ID NO: 28QVTLRESGPA LVKPTQTLTL TCTFSGFSLA PTSSSTKKTQ LQLEHLLLDL QMILNGINNY  60V_(H) KNPKLTAMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV120 IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG WIRQPPGKAL180 EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC ARSMITNWYF240 DVWGAGTTVT VSS 253 SEQ ID NO: 29QMILNGINNY KNPKLTAMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR  60Heavy chainPRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG 120WIRQPPGKAL EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC 180ARSMITNWYF DVWGAGTTVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV 240TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR 300VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV AVSHEDPEVK 360FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALAAPIEK 420TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT 480PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 533SEQ ID NO: 30 KAQLSVGYMH  10 LCDR1 kabat SEQ ID NO: 31 DTSKLAS   7LCDR2 kabat SEQ ID NO: 32 FQGSGYPFT   9 LCDR3 kabat SEQ ID NO: 33 QLSVGY  6 LCDR1 chothia SEQ ID NO: 34 DTS   3 LCDR2 chothia SEQ ID NO: 35GSGYPF   6 LCDR3 chothia SEQ ID NO: 36DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR  60V_(L) FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIK 106SEQ ID NO: 37DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR  60Light chainFSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIKRTVA APSVFIFPPS 120DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 180SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 213 SEQ ID NO: 38QVTLRESGPA LVKPTQTLTL TCTFSGFSLA PTSSSTKKTQ LQLEHLLLDL QMILNGINNY  60Light chainKNPKLTRMLT AKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV 120IVIELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG WIRQPPGKAL 180EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC ARSMITNWYF 240DVWGAGTTVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT 300SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR VEPKSCDKTH 360TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV AVSHEDPEVK FNWYVDGVEV 420HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALAAPIEK TISKAKGQPR 480EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF 540FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 583 SEQ ID NO: 39DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR  60Light chainFSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIKRTVA APSVFIFPPS 120DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 180SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 213

The term “IL-4” (also referred to herein as “IL4”) refers to thecytokine known as interleukin 4, which is produced by Th2 T cells and byeosinophils, basophils, and mast cells. IL-4 regulates thedifferentiation of naïve helper T cells (Th0 cells) to Th2 T cells.Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation byIL-4, Th2 T cells subsequently produce additional IL-4 in a positivefeedback loop. IL-4 also stimulates B cell proliferation and class IIMEW expression, and induces class switching to IgE and IgG₁ expressionfrom B cells. Recombinant human IL-4 suitable for use in the inventionis commercially available from multiple suppliers, includingProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No.CYT-211) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (humanIL-15 recombinant protein, Cat. No. Gibco CTP0043). The amino acidsequence of recombinant human IL-4 suitable for use in the invention isgiven in Table 2 (SEQ ID NO:9).

The term “IL-4” (also referred to herein as “IL4”) refers to thecytokine known as interleukin 4, which is produced by Th2 T cells and byeosinophils, basophils, and mast cells. IL-4 regulates thedifferentiation of naïve helper T cells (Th0 cells) to Th2 T cells.Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation byIL-4, Th2 T cells subsequently produce additional IL-4 in a positivefeedback loop. IL-4 also stimulates B cell proliferation and class IIMEW expression, and induces class switching to IgE and IgG₁ expressionfrom B cells. Recombinant human IL-4 suitable for use in the inventionis commercially available from multiple suppliers, includingProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No.CYT-211) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (humanIL-15 recombinant protein, Cat. No. Gibco CTP0043). The amino acidsequence of recombinant human IL-4 suitable for use in the invention isgiven in Table 2 (SEQ ID NO:5).

The term “IL-7” (also referred to herein as “IL7”) refers to aglycosylated tissue-derived cytokine known as interleukin 7, which maybe obtained from stromal and epithelial cells, as well as from dendriticcells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate thedevelopment of T cells. IL-7 binds to the IL-7 receptor, a heterodimerconsisting of IL-7 receptor alpha and common gamma chain receptor, whichin a series of signals important for T cell development within thethymus and survival within the periphery. Recombinant human IL-7suitable for use in the invention is commercially available frommultiple suppliers, including ProSpec-Tany TechnoGene Ltd., EastBrunswick, N.J., USA (Cat. No. CYT-254) and ThermoFisher Scientific,Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No.Gibco PHC0071). The amino acid sequence of recombinant human IL-7suitable for use in the invention is given in Table 2 (SEQ ID NO:6).

The term “IL-15” (also referred to herein as “IL15”) refers to the Tcell growth factor known as interleukin-15 and includes all forms ofIL-2 including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-15 isdescribed, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, thedisclosure of which is incorporated by reference herein. IL-15 shares βand γ signaling receptor subunits with IL-2. Recombinant human IL-15 isa single, non-glycosylated polypeptide chain containing 114 amino acids(and an N-terminal methionine) with a molecular mass of 12.8 kDa.Recombinant human IL-15 is commercially available from multiplesuppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J.,USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham,Mass., USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). Theamino acid sequence of recombinant human IL-15 suitable for use in theinvention is given in Table 2 (SEQ ID NO:7).

The term “IL-21” (also referred to herein as “IL21”) refers to thepleiotropic cytokine protein known as interleukin-21 and includes allforms of IL-21 including human and mammalian forms, conservative aminoacid substitutions, glycoforms, biosimilars, and variants thereof. IL-21is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014,13, 379-95, the disclosure of which is incorporated by reference herein.IL-21 is primarily produced by natural killer T cells and activatedhuman CD4⁺ T cells. Recombinant human IL-21 is a single,non-glycosylated polypeptide chain containing 132 amino acids with amolecular mass of 15.4 kDa. Recombinant human IL-21 is commerciallyavailable from multiple suppliers, including ProSpec-Tany TechnoGeneLtd., East Brunswick, N.J., USA (Cat. No. CYT-408-b) and ThermoFisherScientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein,Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21suitable for use in the invention is given in Table 2 (SEQ ID NO:8).

When “an anti-tumor effective amount”, “a tumor-inhibiting effectiveamount”, or “therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of infection or metastasis, andcondition of the patient (subject). It can generally be stated that apharmaceutical composition comprising the tumor infiltrating lymphocytes(e.g., secondary TILs or genetically modified cytotoxic lymphocytes)described herein may be administered at a dosage of 10⁴ to 10¹¹ cells/kgbody weight (e.g., 10⁵ to 10⁶, 10⁵ to 10¹⁰, 10⁵ to 10¹¹, 10⁶ to 10¹⁰,10⁶ to 10¹¹, 10⁷ to 10¹¹, 10⁷ to 10¹⁰, 10⁸ to 10¹¹, 10⁸ to 10¹⁰, 10⁹ to10¹¹, or 10⁹ to 10¹⁰ cells/kg body weight), including all integer valueswithin those ranges. Tumor infiltrating lymphocytes (including in somecases, genetically modified cytotoxic lymphocytes) compositions may alsobe administered multiple times at these dosages. The tumor infiltratinglymphocytes (including in some cases, genetically modified cytotoxiclymphocytes) can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

The term “hematological malignancy”, “hematologic malignancy” or termsof correlative meaning refer to mammalian cancers and tumors of thehematopoietic and lymphoid tissues, including but not limited to tissuesof the blood, bone marrow, lymph nodes, and lymphatic system.Hematological malignancies are also referred to as “liquid tumors.”Hematological malignancies include, but are not limited to, acutelymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), smalllymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CIVIL), acute monocytic leukemia (AMoL), Hodgkin'slymphoma, and non-Hodgkin's lymphomas. The term “B cell hematologicalmalignancy” refers to hematological malignancies that affect B cells.

The term “solid tumor” refers to an abnormal mass of tissue that usuallydoes not contain cysts or liquid areas. Solid tumors may be benign ormalignant. The term “solid tumor cancer” refers to malignant,neoplastic, or cancerous solid tumors. Solid tumor cancers include, butare not limited to, sarcomas, carcinomas, and lymphomas, such as cancersof the lung, breast, prostate, colon, rectum, and bladder. The tissuestructure of solid tumors includes interdependent tissue compartmentsincluding the parenchyma (cancer cells) and the supporting stromal cellsin which the cancer cells are dispersed and which may provide asupporting microenvironment.

The term “liquid tumor” refers to an abnormal mass of cells that isfluid in nature. Liquid tumor cancers include, but are not limited to,leukemias, myelomas, and lymphomas, as well as other hematologicalmalignancies. TILs obtained from liquid tumors may also be referred toherein as marrow infiltrating lymphocytes (MILs). TILs obtained fromliquid tumors, including liquid tumors circulating in peripheral blood,may also be referred to herein as PBLs. The terms MIL, TIL, and PBL areused interchangeably herein and differ only based on the tissue typefrom which the cells are derived.

The term “microenvironment,” as used herein, may refer to the solid orhematological tumor microenvironment as a whole or to an individualsubset of cells within the microenvironment. The tumor microenvironment,as used herein, refers to a complex mixture of “cells, soluble factors,signaling molecules, extracellular matrices, and mechanical cues thatpromote neoplastic transformation, support tumor growth and invasion,protect the tumor from host immunity, foster therapeutic resistance, andprovide niches for dominant metastases to thrive,” as described inSwartz, et al., Cancer Res., 2012, 72, 2473. Although tumors expressantigens that should be recognized by T-cells, tumor clearance by theimmune system is rare because of immune suppression by themicroenvironment.

The term “dynamically scheduling,” or “dynamic scheduling” as usedherein, may refer to creating a flexible schedule for events to takeplace based on an outcome or result of one or more subsequent events.Thus, a scheduled date for an event, such as a patient treatment event,may be dynamically changed based on an outcome or a result of one ormore manufacturing steps performed after scheduling the scheduled date,but before the event.

In an embodiment, the invention includes a method of treating a cancerwith a population of TILs, wherein a patient is pre-treated withnon-myeloablative chemotherapy prior to an infusion of TILs according tothe invention. In some embodiments, the population of TILs may beprovided wherein a patient is pre-treated with nonmyeloablativechemotherapy prior to an infusion of TILs according to the presentinvention. In an embodiment, the non-myeloablative chemotherapy iscyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TILinfusion) and fludarabine 25 mg/m²/d for 5 days (days 27 to 23 prior toTIL infusion). In an embodiment, after non-myeloablative chemotherapyand TIL infusion (at day 0) according to the invention, the patientreceives an intravenous infusion of IL-2 intravenously at 720,000 IU/kgevery 8 hours to physiologic tolerance.

Experimental findings indicate that lymphodepletion prior to adoptivetransfer of tumor-specific T lymphocytes plays a key role in enhancingtreatment efficacy by eliminating regulatory T-cells and competingelements of the immune system (“cytokine sinks”). Accordingly, someembodiments of the invention utilize a lymphodepletion step (sometimesalso referred to as “immunosuppressive conditioning”) on the patientprior to the introduction of the rTILs of the invention.

The terms “co-administration,” “co-administering,” “administered incombination with,” “administering in combination with,” “simultaneous,”and “concurrent,” as used herein, encompass administration of two ormore active pharmaceutical ingredients (in a preferred embodiment of thepresent invention, for example, at least one potassium channel agonistin combination with a plurality of TILs) to a subject so that bothactive pharmaceutical ingredients and/or their metabolites are presentin the subject at the same time. Co-administration includes simultaneousadministration in separate compositions, administration at differenttimes in separate compositions, or administration in a composition inwhich two or more active pharmaceutical ingredients are present.Simultaneous administration in separate compositions and administrationin a composition in which both agents are present are preferred.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound or combination of compounds as describedherein that is sufficient to effect the intended application including,but not limited to, disease treatment. A therapeutically effectiveamount may vary depending upon the intended application (in vitro or invivo), or the subject and disease condition being treated (e.g., theweight, age and gender of the subject), the severity of the diseasecondition, or the manner of administration. The term also applies to adose that will induce a particular response in target cells (e.g., thereduction of platelet adhesion and/or cell migration). The specific dosewill vary depending on the particular compounds chosen, the dosingregimen to be followed, whether the compound is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichthe compound is carried.

The terms “treatment”, “treating”, “treat”, and the like, refer toobtaining a desired pharmacologic and/or physiologic effect. The effectmay be prophylactic in terms of completely or partially preventing adisease or symptom thereof and/or may be therapeutic in terms of apartial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its developmentor progression; and (c) relieving the disease, i.e., causing regressionof the disease and/or relieving one or more disease symptoms.“Treatment” is also meant to encompass delivery of an agent in order toprovide for a pharmacologic effect, even in the absence of a disease orcondition. For example, “treatment” encompasses delivery of acomposition that can elicit an immune response or confer immunity in theabsence of a disease condition, e.g., in the case of a vaccine.

The term “heterologous” when used with reference to portions of anucleic acid or protein indicates that the nucleic acid or proteincomprises two or more subsequences that are not found in the samerelationship to each other in nature. For instance, the nucleic acid istypically recombinantly produced, having two or more sequences fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source, orcoding regions from different sources. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The terms “sequence identity,” “percent identity,” and “sequence percentidentity” (or synonyms thereof, e.g., “99% identical”) in the context oftwo or more nucleic acids or polypeptides, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned (introducing gaps, if necessary) for maximumcorrespondence, not considering any conservative amino acidsubstitutions as part of the sequence identity. The percent identity canbe measured using sequence comparison software or algorithms or byvisual inspection. Various algorithms and software are known in the artthat can be used to obtain alignments of amino acid or nucleotidesequences. Suitable programs to determine percent sequence identityinclude for example the BLAST suite of programs available from the U.S.Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be carried using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. ALIGN,ALIGN-2 (Genentech, South San Francisco, Calif.) or MegAlign, availablefrom DNASTAR, are additional publicly available software programs thatcan be used to align sequences. One skilled in the art can determineappropriate parameters for maximal alignment by particular alignmentsoftware. In certain embodiments, the default parameters of thealignment software are used.

As used herein, the term “variant” encompasses but is not limited toproteins, antibodies or fusion proteins which comprise an amino acidsequence which differs from the amino acid sequence of a referenceprotein, antibody or fusion protein by way of one or more substitutions,deletions and/or additions at certain positions within or adjacent tothe amino acid sequence of the reference antibody, protein, or fusionprotein. The variant may comprise one or more conservative substitutionsin its amino acid sequence as compared to the amino acid sequence of areference antibody. Conservative substitutions may involve, e.g., thesubstitution of similarly charged or uncharged amino acids. The variantretains the ability to specifically bind to the antigen of the referenceantibody, protein, or fusion protein. The term variant also includespegylated antibodies or proteins.

By “tumor infiltrating lymphocytes” or “TILs” herein is meant apopulation of cells originally obtained as white blood cells that haveleft the bloodstream of a subject and migrated into a tumor. TILsinclude, but are not limited to, CD8⁺ cytotoxic T-cells (lymphocytes),Th1 and Th17 CD4⁺ T-cells, natural killer cells, dendritic cells and M1macrophages. TILs include both primary and secondary TILs. “PrimaryTILs” are those that are obtained from patient tissue samples asoutlined herein (sometimes referred to as “freshly obtained” or “freshlyisolated”), and “secondary TILs” are any TIL cell populations that havebeen expanded or proliferated as discussed herein, including, but notlimited to bulk TILs, expanded TILs (“REP TILs”) as well as “reREP TILs”as discussed herein. reREP TILs can include for example second expansionTILs or second additional expansion TILs (such as, for example, thosedescribed in Step D of FIG. 2A and/or FIG. 9 , including TILs referredto as reREP TILs).

TILs can generally be defined either biochemically, using cell surfacemarkers, or functionally, by their ability to infiltrate tumors andeffect treatment. TILs can be generally categorized by expressing one ormore of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56,CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively,TILs can be functionally defined by their ability to infiltrate solidtumors upon reintroduction into a patient. TILS may further becharacterized by potency—for example, TILS may be considered potent if,for example, interferon (IFN) release is greater than about 50 pg/mL,greater than about 100 pg/mL, greater than about 150 pg/mL, or greaterthan about 200 pg/mL. TILS may be considered potent if, for example,interferon (IFNγ) release is greater than about 50 pg/mL, greater thanabout 100 pg/mL, greater than about 150 pg/mL, or greater than about 200pg/mL, greater than about 300 pg/mL, greater than about 400 pg/mL,greater than about 500 pg/mL, greater than about 600 pg/mL, greater thanabout 700 pg/mL, greater than about 800 pg/mL, greater than about 900pg/mL, greater than about 1000 pg/mL.

The terms “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” are intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and inert ingredients. The useof such pharmaceutically acceptable carriers or pharmaceuticallyacceptable excipients for active pharmaceutical ingredients is wellknown in the art. Except insofar as any conventional pharmaceuticallyacceptable carrier or pharmaceutically acceptable excipient isincompatible with the active pharmaceutical ingredient, its use in thetherapeutic compositions of the invention is contemplated. Additionalactive pharmaceutical ingredients, such as other drugs, can also beincorporated into the described compositions and methods.

The terms “about” and “approximately” mean within a statisticallymeaningful range of a value. Such a range can be within an order ofmagnitude, preferably within 50%, more preferably within 20%, morepreferably still within 10%, and even more preferably within 5% of agiven value or range. The allowable variation encompassed by the terms“about” or “approximately” depends on the particular system under study,and can be readily appreciated by one of ordinary skill in the art.Moreover, as used herein, the terms “about” and “approximately” meanthat dimensions, sizes, formulations, parameters, shapes and otherquantities and characteristics are not and need not be exact, but may beapproximate and/or larger or smaller, as desired, reflecting tolerances,conversion factors, rounding off, measurement error and the like, andother factors known to those of skill in the art. In general, adimension, size, formulation, parameter, shape or other quantity orcharacteristic is “about” or “approximate” whether or not expresslystated to be such. It is noted that embodiments of very different sizes,shapes and dimensions may employ the described arrangements.

The transitional terms “comprising,” “consisting essentially of,” and“consisting of,” when used in the appended claims, in original andamended form, define the claim scope with respect to what unrecitedadditional claim elements or steps, if any, are excluded from the scopeof the claim(s). The term “comprising” is intended to be inclusive oropen-ended and does not exclude any additional, unrecited element,method, step or material. The term “consisting of” excludes any element,step or material other than those specified in the claim and, in thelatter instance, impurities ordinary associated with the specifiedmaterial(s). The term “consisting essentially of” limits the scope of aclaim to the specified elements, steps or material(s) and those that donot materially affect the basic and novel characteristic(s) of theclaimed invention. All compositions, methods, and kits described hereinthat embody the present invention can, in alternate embodiments, be morespecifically defined by any of the transitional terms “comprising,”“consisting essentially of,” and “consisting of.”

II. TIL Manufacturing Processes (Embodiments of GEN3 Processes)

Without being limited to any particular theory, it is believed that thepriming first expansion that primes an activation of T-cells followed bythe rapid second expansion that boosts the activation of T-cells asdescribed in the methods of the invention allows the preparation ofexpanded T-cells that retain a “younger” phenotype, and as such theexpanded T-cells of the invention are expected to exhibit greatercytotoxicity against cancer cells than T-cells expanded by othermethods. In particular, it is believed that an activation of T-cellsthat is primed by exposure to an anti-CD3 antibody (e.g., OKT-3), IL-2and optionally antigen-presenting cells (APCs) and then boosted bysubsequent exposure to additional anti-CD-3 antibody (e.g., OKT-3), IL-2and APCs as taught by the methods of the invention limits or avoids thematuration of T-cells in culture, yielding a population of T-cells witha less mature phenotype, which T-cells are less exhausted by expansionin culture and exhibit greater cytotoxicity against cancer cells. Insome embodiments, the step of rapid second expansion is split into aplurality of steps to achieve a scaling up of the culture by: (a)performing the rapid second expansion by culturing T-cells in a smallscale culture in a first container, e.g., a G-REX 100MCS container, fora period of about 3 to 4 days, and then (b) effecting the transfer ofthe T-cells in the small scale culture to a second container larger thanthe first container, e.g., a G-REX 500MCS container, and culturing theT-cells from the small scale culture in a larger scale culture in thesecond container for a period of about 4 to 7 days. In some embodiments,the step of rapid expansion is split into a plurality of steps toachieve a scaling out of the culture by: (a) performing the rapid secondexpansion by culturing T-cells in a first small scale culture in a firstcontainer, e.g., a G-REX 100MCS container, for a period of about 3 to 4days, and then (b) effecting the transfer and apportioning of theT-cells from the first small scale culture into and amongst at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20second containers that are equal in size to the first container, whereinin each second container the portion of the T-cells from first smallscale culture transferred to such second container is cultured in asecond small scale culture for a period of about 4 to 7 days. In someembodiments, the step of rapid expansion is split into a plurality ofsteps to achieve a scaling out and scaling up of the culture by: (a)performing the rapid second expansion by culturing T-cells in a smallscale culture in a first container, e.g., a G-REX 100MCS container, fora period of about 3 to 4 days, and then (b) effecting the transfer andapportioning of the T-cells from the small scale culture into andamongst at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 second containers that are larger in size than the firstcontainer, e.g., G-REX 500MCS containers, wherein in each secondcontainer the portion of the T-cells from the small scale culturetransferred to such second container is cultured in a larger scaleculture for a period of about 4 to 7 days. In some embodiments, the stepof rapid expansion is split into a plurality of steps to achieve ascaling out and scaling up of the culture by: (a) performing the rapidsecond expansion by culturing T-cells in a small scale culture in afirst container, e.g., a G-REX 100MCS container, for a period of about 4days, and then (b) effecting the transfer and apportioning of theT-cells from the small scale culture into and amongst 2, 3 or 4 secondcontainers that are larger in size than the first container, e.g., G-REX500MCS containers, wherein in each second container the portion of theT-cells from the small scale culture transferred to such secondcontainer is cultured in a larger scale culture for a period of about 5days.

An exemplary TIL process known as process 3 (also referred to herein asGEN3) containing some of these features is depicted in FIGS. 2A and 2Cand/or FIG. 9 , and some of the advantages of this embodiment of thepresent disclosure over process 2A are described in FIG. 2B. Twoembodiments of process 3 are shown in FIG. 2C. Process 2A or Gen 2 isalso described in U.S. Patent Publication No. 2018/.0280436,incorporated by reference herein in its entirety. Process 3 or Gen 3 isalso described in International Patent Application No.PCT/US2019/059718, incorporated by reference herein in its entirety, aswell as figures and FIGS. 5, 6, 8, 9, 10, and 11 as provided in thepresent application.

In some embodiments, the rapid second expansion is performed after theactivation of T-cells effected by the priming first expansion begins todecrease, abate, decay or subside.

In some embodiments, the rapid second expansion is performed after theactivation of T-cells effected by the priming first expansion hasdecreased by at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

In some embodiments, the rapid second expansion is performed after theactivation of T-cells effected by the priming first expansion hasdecreased by a percentage in the range of at or about 1% to 100%.

In some embodiments, the rapid second expansion is performed after theactivation of T-cells effected by the priming first expansion hasdecreased by a percentage in the range of at or about 1% to 10%, 10% to20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to80%, 80% to 90%, or 90% to 100%.

In some embodiments, the rapid second expansion is performed after theactivation of T-cells effected by the priming first expansion hasdecreased by at least at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

In some embodiments, the rapid second expansion is performed after theactivation of T-cells effected by the priming first expansion hasdecreased by up to at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

In some embodiments, the decrease in the activation of T-cells effectedby the priming first expansion is determined by a reduction in theamount of interferon gamma released by the T-cells in response tostimulation with antigen. A reduction in the amount of interferon gammareleased by the T-cells of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 45%, 50%, 55%, 60%, 65%, 70%, and/or 75% as compared to an initialor control level of interferon gamma is indicative of a decrease in theactivation of the T-cells. A reduction in the amount of interferon gammareleased by the T-cells to less than 200 pg/mL, less than 250 pg/mL,less than 300 pg/mL, less than 350 pg/mL, less than 400 pg/mL, less than450 pg/mL, less than 500 pg/mL, less than 550 pg/mL, less than 600pg/mL, less than 650 pg/mL, less than 700 pg/mL, less than 750 pg/mL,less than 800 pg/mL, less than 850 pg/mL, less than 900 pg/mL, less than950 pg/mL, or less than 1000 pg/mL is indicative of a decrease in theactivation of the T-cells.

In some embodiments, the priming first expansion of T-cells is performedduring a period of up to at or about 7 days or about 8 days.

In some embodiments, the priming first expansion of T-cells is performedduring a period of up to at or about 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, or 8 days.

In some embodiments, the priming first expansion of T-cells is performedduring a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, or 8 days.

In some embodiments, the rapid second expansion of T-cells is performedduring a period of up to at or about 11 days.

In some embodiments, the rapid second expansion of T-cells is performedduring a period of up to at or about 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days.

In some embodiments, the rapid second expansion of T-cells is performedduring a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days or 11 days.

In some embodiments, the priming first expansion of T-cells is performedduring a period of from at or about 1 day to at or about 7 days and therapid second expansion of T-cells is performed during a period of fromat or about 1 day to at or about 11 days.

In some embodiments, the priming first expansion of T-cells is performedduring a period of up to at or about 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, or 8 days and the rapid second expansion ofT-cells is performed during a period of up to at or about 1 day, 2 days,3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11days.

In some embodiments, the priming first expansion of T-cells is performedduring a period of from at or about 1 day to at or about 8 days and therapid second expansion of T-cells is performed during a period of fromat or about 1 day to at or about 9 days.

In some embodiments, the priming first expansion of T-cells is performedduring a period of 8 days and the rapid second expansion of T-cells isperformed during a period of 9 days.

In some embodiments, the priming first expansion of T-cells is performedduring a period of from at or about 1 day to at or about 7 days and therapid second expansion of T-cells is performed during a period of fromat or about 1 day to at or about 9 days.

In some embodiments, the priming first expansion of T-cells is performedduring a period of 7 days and the rapid second expansion of T-cells isperformed during a period of 9 days.

In some embodiments, the T-cells are tumor infiltrating lymphocytes(TILs).

In some embodiments, the T-cells are marrow infiltrating lymphocytes(MILs).

In some embodiments, the T-cells are peripheral blood lymphocytes(PBLs).

In some embodiments, the T-cells are obtained from a donor sufferingfrom a cancer.

In some embodiments, the T-cells are TILs obtained from a tumor excisedfrom a patient suffering from a cancer.

In some embodiments, the T-cells are MILs obtained from bone marrow of apatient suffering from a hematologic malignancy.

In some embodiments, the T-cells are PBLs obtained from peripheral bloodmononuclear cells (PBMCs) from a donor. In some embodiments, the donoris suffering from a cancer. In some embodiments, the cancer is thecancer is selected from the group consisting of melanoma, ovariancancer, endometrial cancer, thyroid cancer, colorectal cancer, cervicalcancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer,breast cancer, cancer caused by human papilloma virus, head and neckcancer (including head and neck squamous cell carcinoma (HNSCC)),glioblastoma (including GBM), gastrointestinal cancer, renal cancer, andrenal cell carcinoma. In some embodiments, the cancer is selected fromthe group consisting of melanoma, ovarian cancer, cervical cancer,non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breastcancer, cancer caused by human papilloma virus, head and neck cancer(including head and neck squamous cell carcinoma (HNSCC)), glioblastoma(including GBM), gastrointestinal cancer, renal cancer, and renal cellcarcinoma. In some embodiments, the donor is suffering from a tumor. Insome embodiments, the tumor is a liquid tumor. In some embodiments, thetumor is a solid tumor. In some embodiments, the donor is suffering froma hematologic malignancy.

In certain aspects of the present disclosure, immune effector cells,e.g., T-cells, can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as FICOLL separation. In one preferred aspect, cells from thecirculating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T-cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In one aspect, the cells collected byapheresis may be washed to remove the plasma fraction and, optionally,to place the cells in an appropriate buffer or media for subsequentprocessing steps. In one embodiment, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. In one aspect, T-cells are isolated from peripheralblood lymphocytes by lysing the red blood cells and depleting themonocytes, for example, by centrifugation through a PERCOLL gradient orby counterflow centrifugal elutriation.

In some embodiments, the T-cells are PBLs separated from whole blood orapheresis product enriched for lymphocytes from a donor. In someembodiments, the donor is suffering from a cancer. In some embodiments,the cancer is the cancer is selected from the group consisting ofmelanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectalcancer, cervical cancer, non-small-cell lung cancer (NSCLC), lungcancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cellcarcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinalcancer, renal cancer, and renal cell carcinoma. In some embodiments, thecancer is selected from the group consisting of melanoma, ovariancancer, cervical cancer, non-small-cell lung cancer (NSCLC), lungcancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cellcarcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinalcancer, renal cancer, and renal cell carcinoma. In some embodiments, thedonor is suffering from a tumor. In some embodiments, the tumor is aliquid tumor. In some embodiments, the tumor is a solid tumor. In someembodiments, the donor is suffering from a hematologic malignancy. Insome embodiments, the PBLs are isolated from whole blood or apheresisproduct enriched for lymphocytes by using positive or negative selectionmethods, i.e., removing the PBLs using a marker(s), e.g., CD3+ CD45+,for T-cell phenotype, or removing non-T-cell phenotype cells, leavingPBLs. In other embodiments, the PBLs are isolated by gradientcentrifugation. Upon isolation of PBLs from donor tissue, the primingfirst expansion of PBLs can be initiated by seeding a suitable number ofisolated PBLs (in some embodiments, approximately 1×10⁷ PBLs) in thepriming first expansion culture according to the priming first expansionstep of any of the methods described herein.

As discussed and generally outlined herein, TILs are taken from apatient sample and manipulated to expand their number prior totransplant into a patient using the TIL expansion process describedherein. In some embodiments, the TILs may be optionally geneticallymanipulated as discussed below. In some embodiments, the TILs may becryopreserved prior to or after expansion. Once thawed, they may also berestimulated to increase their metabolism prior to infusion into apatient.

In some embodiments, the priming first expansion (including processesreferred herein as the pre-Rapid Expansion (Pre-REP), as well asprocesses shown in FIG. 2A as Step B) is shortened to 1 to 8 days andthe rapid second expansion (including processes referred to herein asRapid Expansion Protocol (REP) as well as processes shown in FIG. 2A asStep D) is shortened to 1 to 9 days, as discussed in detail below aswell as in the examples and figures. In some embodiments, the primingfirst expansion (including processes referred herein as the pre-RapidExpansion (Pre-REP), as well as processes shown in FIG. 2A as Step B) isshortened to 1 to 8 days and the rapid second expansion (includingprocesses referred to herein as Rapid Expansion Protocol (REP) as wellas processes shown in FIG. 2A as Step D) is shortened to 1 to 8 days, asdiscussed in detail below as well as in the examples and figures. Insome embodiments, the priming first expansion (including processesreferred herein as the pre-Rapid Expansion (Pre-REP), as well asprocesses shown in FIG. 2A as Step B) is shortened to 1 to 7 days andthe rapid second expansion (including processes referred to herein asRapid Expansion Protocol (REP) as well as processes shown in FIG. 2A asStep D) is shortened to 1 to 9 days, as discussed in detail below aswell as in the examples and figures. In some embodiments, the primingfirst expansion (including processes referred herein as the pre-RapidExpansion (Pre-REP), as well as processes shown in FIG. 2A as Step B) is1 to 7 days and the rapid second expansion (including processes referredto herein as Rapid Expansion Protocol (REP) as well as processes shownin FIG. 2A as Step D is 1 to 10 days, as discussed in detail below aswell as in the examples and figures. In some embodiments, the primingfirst expansion (for example, an expansion described as Step B in FIG.2A) is shortened to 8 days and the rapid second expansion (for example,an expansion as described in Step D in FIG. 2A) is 7 to 9 days. In someembodiments, the priming first expansion (for example, an expansiondescribed as Step B in FIG. 2A) is 8 days and the rapid second expansion(for example, an expansion as described in Step D in FIG. 2A) is 8 to 9days. In some embodiments, the priming first expansion (for example, anexpansion described as Step B in FIG. 2A is shortened to 7 days and therapid second expansion (for example, an expansion as described in Step Din FIG. 2A) is 7 to 8 days. In some embodiments, the priming firstexpansion (for example, an expansion described as Step B in FIG. 2A) isshortened to 8 days and the rapid second expansion (for example, anexpansion as described in Step D in FIG. 2A) is 8 days. In someembodiments, the priming first expansion (for example, an expansiondescribed as Step B in FIG. 2A) is 8 days and the rapid second expansion(for example, an expansion as described in Step D in FIG. 2A) is 9 days.In some embodiments, the priming first expansion (for example, anexpansion described as Step B in FIG. 2A) is 8 days and the rapid secondexpansion (for example, an expansion as described in Step D in FIG. 2A)is 10 days. In some embodiments, the priming first expansion (forexample, an expansion described as Step B in FIG. 2A) is 7 days and therapid second expansion (for example, an expansion as described in Step Din FIG. 2A) is 7 to 10 days. In some embodiments, the priming firstexpansion (for example, an expansion described as Step B in FIG. 2A) is7 days and the rapid second expansion (for example, an expansion asdescribed in Step D in FIG. 2A) is 8 to 10 days. In some embodiments,the priming first expansion (for example, an expansion described as StepB in FIG. 2A) is 7 days and the rapid second expansion (for example, anexpansion as described in Step D in FIG. 2A is 9 to 10 days. In someembodiments, the priming first expansion (for example, an expansiondescribed as Step B in FIG. 2A) is shortened to 7 days and the rapidsecond expansion (for example, an expansion as described in Step D inFIG. 2A) is 7 to 9 days. In some embodiments, the combination of thepriming first expansion and rapid second expansion (for example,expansions described as Step B and Step D in FIG. 2A) is 14-16 days, asdiscussed in detail below and in the examples and figures. Particularly,it is considered that certain embodiments of the present inventioncomprise a priming first expansion step in which TILs are activated byexposure to an anti-CD3 antibody, e.g., OKT-3 in the presence of IL-2 orexposure to an antigen in the presence of at least IL-2 and an anti-CD3antibody e.g., OKT-3. In certain embodiments, the TILs which areactivated in the priming first expansion step as described above are afirst population of TILs i.e., which are a primary cell population.

In some embodiments, the TILs are not stored after the first expansionand prior to the second expansion, and the TILs proceed directly to thesecond expansion (for example, in some embodiments, there is no storageduring the transition from Step B to Step D as shown in FIG. 2A). Insome embodiments, the transition occurs in closed system, as describedherein. In some embodiments, the TILs from the first expansion, thesecond population of TILs, proceeds directly into the second expansionwith no transition period.

In some embodiments, the first expansion, for example, Step B accordingto FIG. 2A, is performed in a closed system bioreactor. In someembodiments, a closed system is employed for the TIL expansion, asdescribed herein. In some embodiments, a single bioreactor is employed.In some embodiments, the single bioreactor employed is for example aG-REX-10 or a G-REX-100. In some embodiments, the closed systembioreactor is a single bioreactor.

The “Step” Designations A, B, C, etc., referred to herein are inreference to the non-limiting example in FIG. 2A and in reference tocertain non-limiting embodiments described herein. The ordering of theSteps below and in FIG. 2A is exemplary and any combination or order ofsteps, as well as additional steps, repetition of steps, and/or omissionof steps is contemplated by the present application and the methodsdisclosed herein.

A. Cell Viability Analyses

A cell viability assay can be performed after the priming firstexpansion (sometimes referred to as the initial bulk expansion), usingstandard assays known in the art. Thus, in certain embodiments, themethod comprises performing a cell viability assay subsequent to thepriming first expansion. In some embodiments, a cell viability assay canbe performed after the second expansion (e.g., after REP), as well asafter the final harvest. For example, a trypan blue exclusion assay canbe done on a sample of the bulk TILs, which selectively labels deadcells and allows a viability assessment. Other assays for use in testingviability can include but are not limited to the Alamar blue assay; andthe MTT assay.

1. Cell Counts, Viability, Flow Cytometry

In some embodiments, cell counts and/or viability are measured. Theexpression of markers such as but not limited CD3, CD4, CD8, and CD56,as well as any other disclosed or described herein, can be measured byflow cytometry with antibodies, for example but not limited to thosecommercially available from BD Bio-sciences (BD Biosciences, San Jose,Calif.) using a FACSCanto™ flow cytometer (BD Biosciences). The cellscan be counted manually using a disposable c-chip hemocytometer (VWR,Batavia, Ill.) and viability can be assessed using any method known inthe art, including but not limited to trypan blue staining. The cellviability can also be assayed based on U.S. Ser. No. 15/863,634,incorporated by reference herein in its entirety. Cell viability canalso be assayed based on U.S. Patent Publication No. 2018/0280436 orInternational Patent Publication No. WO/2018/081473, both of which areincorporate herein in their entireties for all purposes.

In some cases, the bulk TIL population can be cryopreserved immediately,using the protocols discussed below. Alternatively, the bulk TILpopulation can be subjected to REP and then cryopreserved as discussedbelow. Similarly, in the case where genetically modified TILs will beused in therapy, the bulk or REP TIL populations can be subjected togenetic modifications for suitable treatments.

III. TIL Manufacturing Processes (Embodiments of Process 2A)

An exemplary TIL process known as process 2A containing some of thesefeatures is depicted in FIG. 5 , and some of the differences andadvantages of this embodiment of the present invention over process 1Care described in FIG. 6 as well as FIG. 11 . Process 1C is shown forcomparison in FIGS. 6, 7, and 10 . An embodiment of process 2A is shownin FIG. 6 as well as FIGS. 5, 9, 10, and 11 . FIGS. 10 and 11 furtherprovides an exemplary 2A process compared to an exemplary 1C process.

As discussed herein, the present invention can include a step relatingto the restimulation of cryopreserved TILs to increase their metabolicactivity and thus relative health prior to transplant into a patient,and methods of testing said metabolic health. As generally outlinedherein, TILs are generally taken from a patient sample and manipulatedto expand their number prior to transplant into a patient. In someembodiments, the TILs may be optionally genetically manipulated asdiscussed below.

In some embodiments, the TILs may be cryopreserved. Once thawed, theymay also be restimulated to increase their metabolism prior to infusioninto a patient.

In some embodiments, the first expansion (including processes referredto as the preREP as well as processes shown in FIG. 9 as Step A) isshortened to 3 to 14 days and the second expansion (including processesreferred to as the REP as well as processes shown in FIG. 9 as Step B)is shorted to 7 to 14 days, as discussed in detail below as well as inthe examples and figures. In some embodiments, the first expansion (forexample, an expansion described as Step B in FIG. 9 ) is shortened to 11days and the second expansion (for example, an expansion as described inStep D in FIG. 9 ) is shortened to 11 days, as discussed in the Examplesand shown in FIGS. 5, 6, 8, 9, 10, and 11 . In some embodiments, thecombination of the first expansion and second expansion (for example,expansions described as Step B and Step D in FIG. 9 ) is shortened to 22days, as discussed in detail below and in the examples and figures.

The “Step” Designations A, B, C, etc., below are in reference to FIG. 9and in reference to certain embodiments described herein. The orderingof the Steps below and in FIG. 9 is exemplary and any combination ororder of steps, as well as additional steps, repetition of steps, and/oromission of steps is contemplated by the present application and themethods disclosed herein.

A. STEP A: Obtain Patient Tumor Sample

In general, TILs are initially obtained from a patient tumor sample andthen expanded into a larger population for further manipulation asdescribed herein, optionally cryopreserved, restimulated as outlinedherein and optionally evaluated for phenotype and metabolic parametersas an indication of TIL health.

A patient tumor sample may be obtained using methods known in the art,generally via surgical resection, needle biopsy, core biopsy, smallbiopsy, or other means for obtaining a sample that contains a mixture oftumor and TIL cells. In some embodiments, multilesional sampling isused. In some embodiments, surgical resection, needle biopsy, corebiopsy, small biopsy, or other means for obtaining a sample thatcontains a mixture of tumor and TIL cells includes multilesionalsampling (i.e., obtaining samples from one or more tumor cites and/orlocations in the patient, as well as one or more tumors in the samelocation or in close proximity). In general, the tumor sample may befrom any solid tumor, including primary tumors, invasive tumors ormetastatic tumors. The tumor sample may also be a liquid tumor, such asa tumor obtained from a hematological malignancy. The solid tumor may beof lung tissue. In some embodiments, useful TILs are obtained fromnon-small cell lung carcinoma (NSCLC).

Once obtained, the tumor sample is generally fragmented using sharpdissection into small pieces of between 1 to about 8 mm3, with fromabout 2-3 mm3 being particularly useful. In some embodiments, the TILsare cultured from these fragments using enzymatic tumor digests. Suchtumor digests may be produced by incubation in enzymatic media (e.g.,Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10mcg/mL gentamicine, 30 units/mL of DNase and 1.0 mg/mL of collagenase)followed by mechanical dissociation (e.g., using a tissue dissociator).Tumor digests may be produced by placing the tumor in enzymatic mediaand mechanically dissociating the tumor for approximately 1 minute,followed by incubation for 30 minutes at 37° C. in 5% CO2, followed byrepeated cycles of mechanical dissociation and incubation under theforegoing conditions until only small tissue pieces are present. At theend of this process, if the cell suspension contains a large number ofred blood cells or dead cells, a density gradient separation usingFICOLL branched hydrophilic polysaccharide may be performed to removethese cells. Alternative methods known in the art may be used, such asthose described in U.S. Patent Application Publication No. 2012/0244133A1, the disclosure of which is incorporated by reference herein. Any ofthe foregoing methods may be used in any of the embodiments describedherein for methods of expanding TILs or methods treating a cancer.

Tumor dissociating enzyme mixtures can include one or more dissociating(digesting) enzymes such as, but not limited to, collagenase (includingany blend or type of collagenase), Accutase™, Accumax™, hyaluronidase,neutral protease (dispase), chymotrypsin, chymopapain, trypsin,caseinase, elastase, papain, protease type XIV (pronase),deoxyribonuclease I (DNase), trypsin inhibitor, any other dissociatingor proteolytic enzyme, and any combination thereof.

In some embodiments, the dissociating enzymes are reconstituted fromlyophilized enzymes. In some embodiments, lyophilized enzymes arereconstituted in an amount of sterile buffer such as HBSS.

In some instances, collagenase (such as animal free-type 1 collagenase)is reconstituted in 10 mL of sterile HBSS or another buffer. Thelyophilized stock enzyme may be at a concentration of 2892 PZ U/vial. Insome embodiments, collagenase is reconstituted in 5 mL to 15 mL buffer.In some embodiment, after reconstitution the collagenase stock rangesfrom about 100 PZ U/mL-about 400 PZ U/mL, e.g., about 100 PZ U/mL-about400 PZ U/mL, about 100 PZ U/mL-about 350 PZ U/mL, about 100 PZU/mL-about 300 PZ U/mL, about 150 PZ U/mL-about 400 PZ U/mL, about 100PZ U/mL, about 150 PZ U/mL, about 200 PZ U/mL, about 210 PZ U/mL, about220 PZ U/mL, about 230 PZ U/mL, about 240 PZ U/mL, about 250 PZ U/mL,about 260 PZ U/mL, about 270 PZ U/mL, about 280 PZ U/mL, about 289.2 PZU/mL, about 300 PZ U/mL, about 350 PZ U/mL, or about 400 PZ U/mL.

In some embodiments, neutral protease is reconstituted in 1 mL ofsterile HBSS or another buffer. The lyophilized stock enzyme may be at aconcentration of 175 DMC U/vial. In some embodiments, afterreconstitution the neutral protease stock ranges from about 100DMC/mL-about 400 DMC/mL, e.g., about 100 DMC/mL-about 400 DMC/mL, about100 DMC/mL-about 350 DMC/mL, about 100 DMC/mL-about 300 DMC/mL, about150 DMC/mL-about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNAse I is reconstituted in 1 mL of sterile HBSS oranother buffer. The lyophilized stock enzyme was at a concentration of 4KU/vial. In some embodiments, after reconstitution the DNase I stockranges from about 1 KU/mL-10 KU/mL, e.g., about 1 KU/mL, about 2 KU/mL,about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, the stock of enzymes is variable and theconcentrations may need to be determined. In some embodiments, theconcentration of the lyophilized stock can be verified. In someembodiments, the final amount of enzyme added to the digest cocktail isadjusted based on the determined stock concentration.

In some embodiment, the enzyme mixture includes about 10.2-ul of neutralprotease (0.36 DMC U/mL), 21.3 μL of collagenase (1.2 PZ/mL) and 250-ulof DNAse I (200 U/mL) in about 4.7 mL of sterile HBSS.

As indicated above, in some embodiments, the TILs are derived from solidtumors. In some embodiments, the solid tumors are not fragmented. Insome embodiments, the solid tumors are not fragmented and are subjectedto enzymatic digestion as whole tumors. In some embodiments, the tumorsare digested in in an enzyme mixture comprising collagenase, DNase, andhyaluronidase. In some embodiments, the tumors are digested in in anenzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2hours. In some embodiments, the tumors are digested in in an enzymemixture comprising collagenase, DNase, and hyaluronidase for 1-2 hoursat 37° C., 5% CO2. In some embodiments, the tumors are digested in in anenzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2hours at 37° C., 5% CO2 with rotation. In some embodiments, the tumorsare digested overnight with constant rotation. In some embodiments, thetumors are digested overnight at 37° C., 5% CO2 with constant rotation.In some embodiments, the whole tumor is combined with the enzymes toform a tumor digest reaction mixture.

In some embodiments, the tumor is reconstituted with the lyophilizedenzymes in a sterile buffer. In some embodiments, the buffer is sterileHBSS.

In some embodiments, the enzyme mixture comprises collagenase. In someembodiments, the collagenase is collagenase IV. In some embodiments, theworking stock for the collagenase is a 100 mg/mL 10× working stock.

In some embodiments, the enzyme mixture comprises DNAse. In someembodiments, the working stock for the DNAse is a 10,000 IU/mL 10×working stock.

In some embodiments, the enzyme mixture comprises hyaluronidase. In someembodiments, the working stock for the hyaluronidase is a 10-mg/mL 10×working stock.

In some embodiments, the enzyme mixture comprises 10 mg/mL collagenase,1000 IU/mL DNAse, and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture comprises 10 mg/mL collagenase,500 IU/mL DNAse, and 1 mg/mL hyaluronidase.

In general, the harvested cell suspension is called a “primary cellpopulation” or a “freshly harvested” cell population.

In some embodiments, fragmentation includes physical fragmentation,including for example, dissection as well as digestion. In someembodiments, the fragmentation is physical fragmentation. In someembodiments, the fragmentation is dissection. In some embodiments, thefragmentation is by digestion. In some embodiments, TILs can beinitially cultured from enzymatic tumor digests and tumor fragmentsobtained from patients. In an embodiment, TILs can be initially culturedfrom enzymatic tumor digests and tumor fragments obtained from patients.

In some embodiments, where the tumor is a solid tumor, the tumorundergoes physical fragmentation after the tumor sample is obtained in,for example, Step A (as provided in FIG. 1 ). In some embodiments, thefragmentation occurs before cryopreservation. In some embodiments, thefragmentation occurs after cryopreservation. In some embodiments, thefragmentation occurs after obtaining the tumor and in the absence of anycryopreservation. In some embodiments, the tumor is fragmented and 10,20, 30, 40 or more fragments or pieces are placed in each container forthe first expansion. In some embodiments, the tumor is fragmented and 30or 40 fragments or pieces are placed in each container for the firstexpansion. In some embodiments, the tumor is fragmented and 40 fragmentsor pieces are placed in each container for the first expansion. In someembodiments, the multiple fragments comprise about 4 to about 50fragments, wherein each fragment has a volume of about 27 mm3. In someembodiments, the multiple fragments comprise about 30 to about 60fragments with a total volume of about 1300 mm3 to about 1500 mm3. Insome embodiments, the multiple fragments comprise about 50 fragmentswith a total volume of about 1350 mm3. In some embodiments, the multiplefragments comprise about 50 fragments with a total mass of about 1 gramto about 1.5 grams. In some embodiments, the multiple fragments compriseabout 4 fragments.

In some embodiments, the TILs are obtained from tumor fragments. In someembodiments, the tumor fragment is obtained by sharp dissection. In someembodiments, the tumor fragment is between about 1 mm3 and 10 mm3. Insome embodiments, the tumor fragment is between about 1 mm3 and 8 mm3.In some embodiments, the tumor fragment is about 1 mm3. In someembodiments, the tumor fragment is about 2 mm3. In some embodiments, thetumor fragment is about 3 mm3. In some embodiments, the tumor fragmentis about 4 mm3. In some embodiments, the tumor fragment is about 5 mm3.In some embodiments, the tumor fragment is about 6 mm3. In someembodiments, the tumor fragment is about 7 mm3. In some embodiments, thetumor fragment is about 8 mm3. In some embodiments, the tumor fragmentis about 9 mm3. In some embodiments, the tumor fragment is about 10 mm3.In some embodiments, the tumors are 1-4 mm×1-4 mm×1-4 mm. In someembodiments, the tumors are 1 mm×1 mm×1 mm. In some embodiments, thetumors are 2 mm×2 mm×2 mm. In some embodiments, the tumors are 3 mm×3mm×3 mm. In some embodiments, the tumors are 4 mm×4 mm×4 mm.

In some embodiments, the tumors are resected in order to minimize theamount of hemorrhagic, necrotic, and/or fatty tissues on each piece. Insome embodiments, the tumors are resected in order to minimize theamount of hemorrhagic tissue on each piece. In some embodiments, thetumors are resected in order to minimize the amount of necrotic tissueon each piece. In some embodiments, the tumors are resected in order tominimize the amount of fatty tissue on each piece.

In some embodiments, the tumor fragmentation is performed in order tomaintain the tumor internal structure. In some embodiments, the tumorfragmentation is performed without preforming a sawing motion with ascalpel. In some embodiments, the TILs are obtained from tumor digests.In some embodiments, tumor digests were generated by incubation inenzyme media, for example but not limited to RPMI 1640, 2 mM GlutaMAX,10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase, fol¬lowedby mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn,Calif.). After placing the tumor in enzyme media, the tumor can bemechanically dissociated for approx¬imately 1 minute. The solution canthen be incubated for 30 minutes at 37° C. in 5% CO2 and it thenmechanically disrupted again for approximately 1 minute. After beingin¬cubated again for 30 minutes at 37° C. in 5% CO2, the tumor can bemechanically disrupted a third time for approximately 1 minute. In someembodiments, after the third mechanical disruption if large pieces oftissue were present, 1 or 2 additional mechanical dissociations wereapplied to the sample, with or with¬out 30 additional minutes ofincubation at 37° C. in 5% CO2. In some embodiments, at the end of thefinal incubation if the cell suspension contained a large number of redblood cells or dead cells, a density gradient separation using Ficollcan be performed to remove these cells.

In some embodiments, the harvested cell suspension prior to the firstexpansion step is called a “primary cell population” or a “freshlyharvested” cell population.

In some embodiments, cells can be optionally frozen after sample harvestand stored frozen prior to entry into the expansion described in Step B,which is described in further detail below, as well as exemplified inFIG. 9 .

1. Pleural Effusion T-Cells and TILs

In some embodiments, the sample is a pleural fluid sample. In someembodiments, the source of the T-cells TILs for expansion according tothe processes described herein is a pleural fluid sample. In someembodiments, the sample is a pleural effusion derived sample. In someembodiments, the source of the T-cells or TILs for expansion accordingto the processes described herein is a pleural effusion derived sample.See, for example, methods described in U.S. Patent Publication US2014/0295426, incorporated herein by reference in its entirety for allpurposes.

In some embodiments, any pleural fluid or pleural effusion suspected ofand/or containing TILs can be employed. Such a sample may be derivedfrom a primary or metastatic lung cancer, such as NSCLC or SCLC. In someembodiments, the sample may be secondary metastatic cancer cells whichoriginated from another organ, e.g., breast, ovary, colon or prostate.In some embodiments, the sample for use in the expansion methodsdescribed herein is a pleural exudate. In some embodiments, the samplefor use in the expansion methods described herein is a pleuraltransudate. Other biological samples may include other serous fluidscontaining TILs, including, e.g., ascites fluid from the abdomen orpancreatic cyst fluid. Ascites fluid and pleural fluids involve verysimilar chemical systems; both the abdomen and lung have mesotheliallines and fluid forms in the pleural space and abdominal spaces in thesame matter in malignancies and such fluids in some embodiments containTILs. In some embodiments, wherein the disclosure exemplifies pleuralfluid, the same methods may be performed with similar results usingascites or other cyst fluids containing TILs.

In some embodiments, the pleural fluid is in unprocessed form, directlyas removed from the patient. In some embodiments, the unprocessedpleural fluid is placed in a standard blood collection tube, such as anEDTA or Heparin tube, prior to the contacting step. In some embodiments,the unprocessed pleural fluid is placed in a standard CellSave® tube(Veridex) prior to the contacting step. In some embodiments, the sampleis placed in the CellSave tube immediately after collection from thepatient to avoid a decrease in the number of viable TILs. The number ofviable TILs can decrease to a significant extent within 24 hours, ifleft in the untreated pleural fluid, even at 4° C. In some embodiments,the sample is placed in the appropriate collection tube within 1 hour, 5hours, 10 hours, 15 hours, or up to 24 hours after removal from thepatient. In some embodiments, the sample is placed in the appropriatecollection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24hours after removal from the patient at 4° C.

In some embodiments, the pleural fluid sample from the chosen subjectmay be diluted. In one embodiment, the dilution is 1:10 pleural fluid todiluent. In another embodiment, the dilution is 1:9 pleural fluid todiluent. In another embodiment, the dilution is 1:8 pleural fluid todiluent. In another embodiment, the dilution is 1:5 pleural fluid todiluent. In another embodiment, the dilution is 1:2 pleural fluid todiluent. In another embodiment, the dilution is 1:1 pleural fluid todiluent. In some embodiments, diluents include saline, phosphatebuffered saline, another buffer or a physiologically acceptable diluent.In some embodiments, the sample is placed in the CellSave tubeimmediately after collection from the patient and dilution to avoid adecrease in the viable TILs, which may occur to a significant extentwithin 24-48 hours, if left in the untreated pleural fluid, even at 4°C. In some embodiments, the pleural fluid sample is placed in theappropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours,24 hours, 36 hours, up to 48 hours after removal from the patient, anddilution. In some embodiments, the pleural fluid sample is placed in theappropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours,24 hours, 36 hours, up to 48 hours after removal from the patient, anddilution at 4° C.

In still another embodiment, pleural fluid samples are concentrated byconventional means prior further processing steps. In some embodiments,this pre-treatment of the pleural fluid is preferable in circumstancesin which the pleural fluid must be cryopreserved for shipment to alaboratory performing the method or for later analysis (e.g., later than24-48 hours post-collection). In some embodiments, the pleural fluidsample is prepared by centrifuging the pleural fluid sample after itswithdrawal from the subject and resuspending the centrifugate or pelletin buffer. In some embodiments, the pleural fluid sample is subjected tomultiple centrifugations and resuspensions, before it is cryopreservedfor transport or later analysis and/or processing.

In some embodiments, pleural fluid samples are concentrated prior tofurther processing steps by using a filtration method. In someembodiments, the pleural fluid sample used in the contacting step isprepared by filtering the fluid through a filter containing a known andessentially uniform pore size that allows for passage of the pleuralfluid through the membrane but retains the tumor cells. In someembodiments, the diameter of the pores in the membrane may be at least 4In another embodiment the pore diameter may be 5 μM or more, and inother embodiment, any of 6, 7, 8, 9, or 10 After filtration, the cells,including TILs, retained by the membrane may be rinsed off the membraneinto a suitable physiologically acceptable buffer. Cells, includingTILs, concentrated in this way may then be used in the contacting stepof the method.

In some embodiments, pleural fluid sample (including, for example, theuntreated pleural fluid), diluted pleural fluid, or the resuspended cellpellet, is contacted with a lytic reagent that differentially lysesnon-nucleated red blood cells present in the sample. In someembodiments, this step is performed prior to further processing steps incircumstances in which the pleural fluid contains substantial numbers ofRBCs. Suitable lysing reagents include a single lytic reagent or a lyticreagent and a quench reagent, or a lytic agent, a quench reagent and afixation reagent. Suitable lytic systems are marketed commercially andinclude the BD Pharm Lyse™ system (Becton Dickenson). Other lyticsystems include the Versalyse™ system, the FACSlyse™ system (BectonDickenson), the Immunoprep™ system or Erythrolyse II system (BeckmanCoulter, Inc.), or an ammonium chloride system. In some embodiments, thelytic reagent can vary with the primary requirements being efficientlysis of the red blood cells, and the conservation of the TILs andphenotypic properties of the TILs in the pleural fluid. In addition toemploying a single reagent for lysis, the lytic systems useful inmethods described herein can include a second reagent, e.g., one thatquenches or retards the effect of the lytic reagent during the remainingsteps of the method, e.g., Stabilyse™ reagent (Beckman Coulter, Inc.). Aconventional fixation reagent may also be employed depending upon thechoice of lytic reagents or the preferred implementation of the method.

In some embodiments, the pleural fluid sample, unprocessed, diluted ormultiply centrifuged or processed as described herein above iscryopreserved at a temperature of about −140° C. prior to being furtherprocessed and/or expanded as provided herein.

B. STEP B: First Expansion

1. Young TILs

In some embodiments, the present methods provide for obtaining youngTILs, which are capable of increased replication cycles uponadministration to a subject/patient and as such may provide additionaltherapeutic benefits over older TILs (i.e., TILs which have furtherundergone more rounds of replication prior to administration to asubject/patient). Features of young TILs have been described in theliterature, for example Donia, at al., Scandinavian Journal ofImmunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res,16:6122-6131 (2010); Huang et al., J Immunother, 28(3):258-267 (2005);Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., JImmunother 32:415-423 (2009); Robbins, et al., J Immunol 2004;173:7125-7130; Shen et al., J Immunother, 30:123-129 (2007); Zhou, etal., J Immunother, 28:53-62 (2005); and Tran, et al., J Immunother,31:742-751 (2008), all of which are incorporated herein by reference intheir entireties.

The diverse antigen receptors of T and B lymphocytes are produced bysomatic recombination of a limited, but large number of gene segments.These gene segments: V (variable), D (diversity), J (joining), and C(constant), determine the binding specificity and downstreamapplications of immunoglobulins and T-cell receptors (TCRs). The presentinvention provides a method for generating TILs which exhibit andincrease the T-cell repertoire diversity. In some embodiments, the TILsobtained by the present method exhibit an increase in the T-cellrepertoire diversity. In some embodiments, the TILs obtained by thepresent method exhibit an increase in the T-cell repertoire diversity ascompared to freshly harvested TILs and/or TILs prepared using othermethods than those provide herein including for example, methods otherthan those embodied in FIG. 9 . In some embodiments, the TILs obtainedby the present method exhibit an increase in the T-cell repertoirediversity as compared to freshly harvested TILs and/or TILs preparedusing methods referred to as process 1C, as exemplified in FIG. 10 . Insome embodiments, the TILs obtained in the first expansion exhibit anincrease in the T-cell repertoire diversity. In some embodiments, theincrease in diversity is an increase in the immunoglobulin diversityand/or the T-cell receptor diversity. In some embodiments, the diversityis in the immunoglobulin is in the immunoglobulin heavy chain. In someembodiments, the diversity is in the immunoglobulin is in theimmunoglobulin light chain. In some embodiments, the diversity is in theT-cell receptor. In some embodiments, the diversity is in one of theT-cell receptors selected from the group consisting of alpha, beta,gamma, and delta receptors. In some embodiments, there is an increase inthe expression of T-cell receptor (TCR) alpha and/or beta. In someembodiments, there is an increase in the expression of T-cell receptor(TCR) alpha. In some embodiments, there is an increase in the expressionof T-cell receptor (TCR) beta. In some embodiments, there is an increasein the expression of TCRab (i.e., TCRα/β).

After dissection or digestion of tumor fragments, for example such asdescribed in Step A of FIG. 9 , the resulting cells are cultured inserum containing IL-2 under conditions that favor the growth of TILsover tumor and other cells. In some embodiments, the tumor digests areincubated in 2 mL wells in media comprising inactivated human AB serumwith 6000 IU/mL of IL-2. This primary cell population is cultured for aperiod of days, generally from 3 to 14 days, resulting in a bulk TILpopulation, generally about 1×10⁸ bulk TIL cells. In some embodiments,this primary cell population is cultured for a period of 7 to 14 days,resulting in a bulk TIL population, generally about 1×10⁸ bulk TILcells. In some embodiments, this primary cell population is cultured fora period of 10 to 14 days, resulting in a bulk TIL population, generallyabout 1×10⁸ bulk TIL cells. In some embodiments, this primary cellpopulation is cultured for a period of about 11 days, resulting in abulk TIL population, generally about 1×10⁸ bulk TIL cells.

In a preferred embodiment, expansion of TILs may be performed using aninitial bulk TIL expansion step (for example such as those described inStep B of FIG. 9 , which can include processes referred to as pre-REP)as described below and herein, followed by a second expansion (Step D,including processes referred to as rapid expansion protocol (REP) steps)as described below under Step D and herein, followed by optionalcryopreservation, and followed by a second Step D (including processesreferred to as restimulation REP steps) as described below and herein.The TILs obtained from this process may be optionally characterized forphenotypic characteristics and metabolic parameters as described herein.

In embodiments where TIL cultures are initiated in 24-well plates, forexample, using Costar 24-well cell culture cluster, flat bottom (CorningIncorporated, Corning, N.Y., each well can be seeded with 1×10⁶ tumordigest cells or one tumor fragment in 2 mL of complete medium (CM) withIL-2 (6000 IU/mL; Chiron Corp., Emeryville, Calif.). In someembodiments, the tumor fragment is between about 1 mm³ and 10 mm³.

In some embodiments, the first expansion culture medium is referred toas “CM”, an abbreviation for culture media. In some embodiments, CM forStep B consists of RPMI 1640 with GlutaMAX, supplemented with 10% humanAB serum, 25 mM HEPES, and 10 mg/mL gentamicin. In embodiments wherecultures are initiated in gas-permeable flasks with a 40 mL capacity anda 10 cm² gas-permeable silicon bottom (for example, G-Rex10; Wilson WolfManufacturing, New Brighton, Minn.) (FIG. 1 ), each flask was loadedwith 10-40×10⁶ viable tumor digest cells or 5-30 tumor fragments in10-40 mL of CM with IL-2. Both the G-Rex10 and 24-well plates wereincubated in a humidified incubator at 37° C. in 5% CO2 and 5 days afterculture initiation, half the media was removed and replaced with freshCM and IL-2 and after day 5, half the media was changed every 2-3 days.

After preparation of the tumor fragments, the resulting cells (i.e.,fragments) are cultured in serum containing IL-2 under conditions thatfavor the growth of TILs over tumor and other cells. In someembodiments, the tumor digests are incubated in 2 mL wells in mediacomprising inactivated human AB serum (or, in some cases, as outlinedherein, in the presence of aAPC cell population) with 6000 IU/mL ofIL-2. This primary cell population is cultured for a period of days,generally from 10 to 14 days, resulting in a bulk TIL population,generally about 1×10⁸ bulk TIL cells. In some embodiments, the growthmedia during the first expansion comprises IL-2 or a variant thereof. Insome embodiments, the IL is recombinant human IL-2 (rhIL-2). In someembodiments the IL-2 stock solution has a specific activity of 20-30×10⁶IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has aspecific activity of 20×10⁶ IU/mg for a 1 mg vial. In some embodimentsthe IL-2 stock solution has a specific activity of 25×10⁶ IU/mg for a 1mg vial. In some embodiments the IL-2 stock solution has a specificactivity of 30×10⁶ IU/mg for a 1 mg vial. In some embodiments, the IL-2stock solution has a final concentration of 4-8×10⁶ IU/mg of IL-2. Insome embodiments, the IL-2 stock solution has a final concentration of5-7×10⁶ IU/mg of IL-2. In some embodiments, the IL-2 stock solution hasa final concentration of 6×10⁶ IU/mg of IL-2. In some embodiments, theIL-2 stock solution is prepare as described in Example 4. In someembodiments, the first expansion culture media comprises about 10,000IU/mL of IL-2, about 9,000 IU/mL of IL-2, about 8,000 IU/mL of IL-2,about 7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000 IU/mLof IL-2. In some embodiments, the first expansion culture mediacomprises about 9,000 IU/mL of IL-2 to about 5,000 IU/mL of IL-2. Insome embodiments, the first expansion culture media comprises about8,000 IU/mL of IL-2 to about 6,000 IU/mL of IL-2. In some embodiments,the first expansion culture media comprises about 7,000 IU/mL of IL-2 toabout 6,000 IU/mL of IL-2. In some embodiments, the first expansionculture media comprises about 6,000 IU/mL of IL-2. In an embodiment, thecell culture medium further comprises IL-2. In some embodiments, thecell culture medium comprises about 3000 IU/mL of IL-2. In anembodiment, the cell culture medium further comprises IL-2. In apreferred embodiment, the cell culture medium comprises about 3000 IU/mLof IL-2. In an embodiment, the cell culture medium comprises about 1000IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment,the cell culture medium comprises between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or about 8000 IU/mL of IL-2.

In some embodiments, first expansion culture media comprises about 500IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15. In some embodiments, the first expansion culture mediacomprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In someembodiments, the first expansion culture media comprises about 400 IU/mLof IL-15 to about 100 IU/mL of IL-15. In some embodiments, the firstexpansion culture media comprises about 300 IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the first expansion culture mediacomprises about 200 IU/mL of IL-15. In some embodiments, the cellculture medium comprises about 180 IU/mL of IL-15. In an embodiment, thecell culture medium further comprises IL-15. In a preferred embodiment,the cell culture medium comprises about 180 IU/mL of IL-15.

In some embodiments, first expansion culture media comprises about 20IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, orabout 0.5 IU/mL of IL-21. In some embodiments, the first expansionculture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL ofIL-21. In some embodiments, the first expansion culture media comprisesabout 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In someembodiments, the first expansion culture media comprises about 12 IU/mLof IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the firstexpansion culture media comprises about 10 IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the first expansion culture mediacomprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In someembodiments, the first expansion culture media comprises about 2 IU/mLof IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL of IL-21. In some embodiments, the cell culture medium comprisesabout 0.5 IU/mL of IL-21. In an embodiment, the cell culture mediumfurther comprises IL-21. In a preferred embodiment, the cell culturemedium comprises about 1 IU/mL of IL-21.

In an embodiment, the cell culture medium comprises OKT-3 antibody. Insome embodiments, the cell culture medium comprises about 30 ng/mL ofOKT-3 antibody. In an embodiment, the cell culture medium comprisesabout 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL,about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1μg/mL of OKT-3 antibody. In an embodiment, the cell culture mediumcomprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL,between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody. In someembodiments, the cell culture medium does not comprise OKT-3 antibody.In some embodiments, the OKT-3 antibody is muromonab.

In some embodiments, the cell culture medium comprises one or moreTNFRSF agonists in a cell culture medium. In some embodiments, theTNFRSF agonist comprises a 4-1BB agonist. In some embodiments, theTNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selectedfrom the group consisting of urelumab, utomilumab, EU-101, a fusionprotein, and fragments, derivatives, variants, biosimilars, andcombinations thereof. In some embodiments, the TNFRSF agonist is addedat a concentration sufficient to achieve a concentration in the cellculture medium of between 0.1 μg/mL and 100 μg/mL. In some embodiments,the TNFRSF agonist is added at a concentration sufficient to achieve aconcentration in the cell culture medium of between 20 μg/mL and 40μg/mL.

In some embodiments, in addition to one or more TNFRSF agonists, thecell culture medium further comprises IL-2 at an initial concentrationof about 3000 IU/mL and OKT-3 antibody at an initial concentration ofabout 30 ng/mL, and wherein the one or more TNFRSF agonists comprises a4-1BB agonist.

In some embodiments, the first expansion culture medium is referred toas “CM”, an abbreviation for culture media. In some embodiments, it isreferred to as CM1 (culture medium 1). In some embodiments, CM consistsof RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mMHEPES, and 10 mg/mL gentamicin. In embodiments where cultures areinitiated in gas-permeable flasks with a 40 mL capacity and a 10 cm²gas-permeable silicon bottom (for example, G-Rex10; Wilson WolfManufacturing, New Brighton, Minn.) (FIG. 1 ), each flask was loadedwith 10-40×10⁶ viable tumor digest cells or 5-30 tumor fragments in10-40 mL of CM with IL-2. Both the G-Rex10 and 24-well plates wereincubated in a humidified incubator at 37° C. in 5% CO2 and 5 days afterculture initiation, half the media was removed and replaced with freshCM and IL-2 and after day 5, half the media was changed every 2-3 days.In some embodiments, the CM is the CM1 described in the Examples, see,Example 5. In some embodiments, the first expansion occurs in an initialcell culture medium or a first cell culture medium. In some embodiments,the initial cell culture medium or the first cell culture mediumcomprises IL-2.

In some embodiments, the first expansion (including processes such asfor example those described in Step B of FIG. 9 , which can includethose sometimes referred to as the pre-REP) process is shortened to 3-14days, as discussed in the examples and figures. In some embodiments, thefirst expansion (including processes such as for example those describedin Step B of FIG. 9 , which can include those sometimes referred to asthe pre-REP) is shortened to 7 to 14 days, as shown in FIGS. 5, 6, 8, 9,10, and 11 , as well as including for example, an expansion as describedin Step B of FIG. 9 . In some embodiments, the first expansion of Step Bis shortened to 10-14 days, as shown in FIGS. 5, 6, 8, 9, 10, and 11 .In some embodiments, the first expansion is shortened to 11 days, asshown in FIGS. 5, 6, 8, 9, 10, and 11 , as well as including forexample, an expansion as described in Step B of FIG. 9 .

In some embodiments, the first TIL expansion can proceed for 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, or 14 days. In some embodiments, the firstTIL expansion can proceed for 1 day to 14 days. In some embodiments, thefirst TIL expansion can proceed for 2 days to 14 days. In someembodiments, the first TIL expansion can proceed for 3 days to 14 days.In some embodiments, the first TIL expansion can proceed for 4 days to14 days. In some embodiments, the first TIL expansion can proceed for 5days to 14 days. In some embodiments, the first TIL expansion canproceed for 6 days to 14 days. In some embodiments, the first TILexpansion can proceed for 7 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 8 days to 14 days. In someembodiments, the first TIL expansion can proceed for 9 days to 14 days.In some embodiments, the first TIL expansion can proceed for 10 days to14 days. In some embodiments, the first TIL expansion can proceed for 11days to 14 days. In some embodiments, the first TIL expansion canproceed for 12 days to 14 days. In some embodiments, the first TILexpansion can proceed for 13 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 14 days. In some embodiments, thefirst TIL expansion can proceed for 1 day to 11 days. In someembodiments, the first TIL expansion can proceed for 2 days to 11 days.In some embodiments, the first TIL expansion can proceed for 3 days to11 days. In some embodiments, the first TIL expansion can proceed for 4days to 11 days. In some embodiments, the first TIL expansion canproceed for 5 days to 11 days. In some embodiments, the first TILexpansion can proceed for 6 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 7 days to 11 days. In someembodiments, the first TIL expansion can proceed for 8 days to 11 days.In some embodiments, the first TIL expansion can proceed for 9 days to11 days. In some embodiments, the first TIL expansion can proceed for 10days to 11 days. In some embodiments, the first TIL expansion canproceed for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the first expansion. In someembodiments, IL-2, IL-7, IL-15, and/or IL-21 as well as any combinationsthereof can be included during the first expansion, including forexample during a Step B processes according to FIG. 9 , as well asdescribed herein. In some embodiments, a combination of IL-2, IL-15, andIL-21 are employed as a combination during the first expansion. In someembodiments, IL-2, IL-15, and IL-21 as well as any combinations thereofcan be included during Step B processes according to FIG. 9 and asdescribed herein.

In some embodiments, the first expansion (including processes referredto as the pre-REP; for example, Step B according to FIG. 9 ) process isshortened to 3 to 14 days, as discussed in the figures. In someembodiments, the first expansion of Step B is shortened to 7 to 14 days,as shown in FIGS. 5, 6, 8, 9, 10, and 11 . In some embodiments, thefirst expansion of Step B is shortened to 10 to 14 days, as shown inFIGS. 5, 6, 8, 9, 10, and 11 . In some embodiments, the first expansionis shortened to 11 days, as shown in FIGS. 5, 6, 8, 9, 10, and 11 .

In some embodiments, the first expansion, for example, Step B accordingto FIG. 9 , is performed in a closed system bioreactor. In someembodiments, a closed system is employed for the TIL expansion, asdescribed herein. In some embodiments, a single bioreactor is employed.In some embodiments, the single bioreactor employed is for example aG-REX-10 or a G-REX-100. In some embodiments, the closed systembioreactor is a single bioreactor.

1. Cytokines and Other Additives

The expansion methods described herein generally use culture media withhigh doses of a cytokine, in particular IL-2, as is known in the art.

Alternatively, using combinations of cytokines for the rapid expansionand or second expansion of TILs is additionally possible, withcombinations of two or more of IL-2, IL-15 and IL-21 as is described inU.S. Patent Application Publication No. US 2017/0107490 A1, thedisclosure of which is incorporated by reference herein. Thus, possiblecombinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 andIL-2, or IL-15 and IL-21, with the latter finding particular use in manyembodiments. The use of combinations of cytokines specifically favorsthe generation of lymphocytes, and in particular T-cells as describedtherein.

In an embodiment, Step B may also include the addition of OKT-3 antibodyor muromonab to the culture media, as described elsewhere herein. In anembodiment, Step B may also include the addition of a 4-1BB agonist tothe culture media, as described elsewhere herein. In an embodiment, StepB may also include the addition of an OX-40 agonist to the culturemedia, as described elsewhere herein. In other embodiments, additivessuch as peroxisome proliferator-activated receptor gamma coactivatorI-alpha agonists, including proliferator-activated receptor (PPAR)-gammaagonists such as a thiazolidinedione compound, may be used in theculture media during Step B, as described in U.S. Patent ApplicationPublication No. US 2019/0307796 A1, the disclosure of which isincorporated by reference herein.

C. STEP C: First Expansion to Second Expansion Transition

In some cases, the bulk TIL population obtained from the firstexpansion, including for example the TIL population obtained from forexample, Step B as indicated in FIG. 9 , can be cryopreservedimmediately, using the protocols discussed herein below. Alternatively,the TIL population obtained from the first expansion, referred to as thesecond TIL population, can be subjected to a second expansion (which caninclude expansions sometimes referred to as REP) and then cryopreservedas discussed below. Similarly, in the case where genetically modifiedTILs will be used in therapy, the first TIL population (sometimesreferred to as the bulk TIL population) or the second TIL population(which can in some embodiments include populations referred to as theREP TIL populations) can be subjected to genetic modifications forsuitable treatments prior to expansion or after the first expansion andprior to the second expansion.

In some embodiments, the TILs obtained from the first expansion (forexample, from Step B as indicated in FIG. 9 ) are stored untilphenotyped for selection. In some embodiments, the TILs obtained fromthe first expansion (for example, from Step B as indicated in FIG. 9 )are not stored and proceed directly to the second expansion. In someembodiments, the TILs obtained from the first expansion are notcryopreserved after the first expansion and prior to the secondexpansion. In some embodiments, the transition from the first expansionto the second expansion occurs at about 3 days, 4, days, 5 days, 6 days,7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs at about 3 days to 14days from when fragmentation occurs. In some embodiments, the transitionfrom the first expansion to the second expansion occurs at about 4 daysto 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs atabout 4 days to 10 days from when fragmentation occurs. In someembodiments, the transition from the first expansion to the secondexpansion occurs at about 7 days to 14 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs at about 14 days from when fragmentationoccurs.

In some embodiments, the transition from the first expansion to thesecond expansion occurs at 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14days from when fragmentation occurs. In some embodiments, the transitionfrom the first expansion to the second expansion occurs 1 day to 14 daysfrom when fragmentation occurs. In some embodiments, the first TILexpansion can proceed for 2 days to 14 days. In some embodiments, thetransition from the first expansion to the second expansion occurs 3days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 4days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 5days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 6days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 7days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 8days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 9days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 10days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 11days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 12days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 13days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 14days from when fragmentation occurs. In some embodiments, the transitionfrom the first expansion to the second expansion occurs 1 day to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 2 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 3 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 4 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 5 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 6 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 7 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 8 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 9 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 10 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 11 days from whenfragmentation occurs.

In some embodiments, the TILs are not stored after the first expansionand prior to the second expansion, and the TILs proceed directly to thesecond expansion (for example, in some embodiments, there is no storageduring the transition from Step B to Step D as shown in FIG. 9 ). Insome embodiments, the transition occurs in closed system, as describedherein. In some embodiments, the TILs from the first expansion, thesecond population of TILs, proceeds directly into the second expansionwith no transition period.

In some embodiments, the transition from the first expansion to thesecond expansion, for example, Step C according to FIG. 9 , is performedin a closed system bioreactor. In some embodiments, a closed system isemployed for the TIL expansion, as described herein. In someembodiments, a single bioreactor is employed. In some embodiments, thesingle bioreactor employed is for example a G-REX-10 or a G-REX-100. Insome embodiments, the closed system bioreactor is a single bioreactor.

D. STEP D: Second Expansion

In some embodiments, the TIL cell population is expanded in number afterharvest and initial bulk processing for example, after Step A and StepB, and the transition referred to as Step C, as indicated in FIG. 9 ).This further expansion is referred to herein as the second expansion,which can include expansion processes generally referred to in the artas a rapid expansion process (REP; as well as processes as indicated inStep D of FIG. 9 ). The second expansion is generally accomplished usinga culture media comprising a number of components, including feedercells, a cytokine source, and an anti-CD3 antibody, in a gas-permeablecontainer.

In some embodiments, the second expansion or second TIL expansion (whichcan include expansions sometimes referred to as REP; as well asprocesses as indicated in Step D of FIG. 9 ) of TIL can be performedusing any TIL flasks or containers known by those of skill in the art.In some embodiments, the second TIL expansion can proceed for 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In someembodiments, the second TIL expansion can proceed for about 7 days toabout 14 days. In some embodiments, the second TIL expansion can proceedfor about 8 days to about 14 days. In some embodiments, the second TILexpansion can proceed for about 9 days to about 14 days. In someembodiments, the second TIL expansion can proceed for about 10 days toabout 14 days. In some embodiments, the second TIL expansion can proceedfor about 11 days to about 14 days. In some embodiments, the second TILexpansion can proceed for about 12 days to about 14 days. In someembodiments, the second TIL expansion can proceed for about 13 days toabout 14 days. In some embodiments, the second TIL expansion can proceedfor about 14 days.

In an embodiment, the second expansion can be performed in a gaspermeable container using the methods of the present disclosure(including for example, expansions referred to as REP; as well asprocesses as indicated in Step D of FIG. 9 ). For example, TILs can berapidly expanded using non-specific T-cell receptor stimulation in thepresence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Thenon-specific T-cell receptor stimulus can include, for example, ananti-CD3 antibody, such as about 30 ng/ml of OKT3, a mouse monoclonalanti-CD3 antibody (commercially available from Ortho-McNeil, Raritan,N.J. or Miltenyi Biotech, Auburn, Calif.) or UHCT-1 (commerciallyavailable from BioLegend, San Diego, Calif., USA). TILs can be expandedto induce further stimulation of the TILs in vitro by including one ormore antigens during the second expansion, including antigenic portionsthereof, such as epitope(s), of the cancer, which can be optionallyexpressed from a vector, such as a human leukocyte antigen A2 (HLA-A2)binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217(210M), optionally in the presence of a T-cell growth factor, such as300 IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g.,NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, andVEGFR2, or antigenic portions thereof. TIL may also be rapidly expandedby re-stimulation with the same antigen(s) of the cancer pulsed ontoHLA-A2-expressing antigen-presenting cells. Alternatively, the TILs canbe further restimulated with, e.g., example, irradiated, autologouslymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.In some embodiments, the re-stimulation occurs as part of the secondexpansion. In some embodiments, the second expansion occurs in thepresence of irradiated, autologous lymphocytes or with irradiatedHLA-A2+ allogeneic lymphocytes and IL-2.

In an embodiment, the cell culture medium further comprises IL-2. In asome embodiments, the cell culture medium comprises about 3000 IU/mL ofIL-2. In an embodiment, the cell culture medium comprises about 1000IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment,the cell culture medium comprises between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.

In an embodiment, the cell culture medium comprises OKT3 antibody. In asome embodiments, the cell culture medium comprises about 30 ng/mL ofOKT3 antibody. In an embodiment, the cell culture medium comprises about0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL,about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL,about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL ofOKT3 antibody. In an embodiment, the cell culture medium comprisesbetween 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL,and between 50 ng/mL and 100 ng/mL of OKT3 antibody.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the second expansion. In someembodiments, IL-2, IL-7, IL-15, and/or IL-21 as well as any combinationsthereof can be included during the second expansion, including forexample during a Step D processes according to FIG. 9 , as well asdescribed herein. In some embodiments, a combination of IL-2, IL-15, andIL-21 are employed as a combination during the second expansion. In someembodiments, IL-2, IL-15, and IL-21 as well as any combinations thereofcan be included during Step D processes according to FIG. 9 and asdescribed herein.

In some embodiments, the second expansion can be conducted in asupplemented cell culture medium comprising IL-2, OKT-3, andantigen-presenting feeder cells. In some embodiments, the secondexpansion occurs in a supplemented cell culture medium. In someembodiments, the supplemented cell culture medium comprises IL-2, OKT-3,and antigen-presenting feeder cells. In some embodiments, the secondcell culture medium comprises IL-2, OKT-3, and antigen-presenting cells(APCs; also referred to as antigen-presenting feeder cells). In someembodiments, the second expansion occurs in a cell culture mediumcomprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e.,antigen presenting cells).

In some embodiments, the second expansion culture media comprises about500 IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15. In some embodiments, the second expansion culture mediacomprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In someembodiments, the second expansion culture media comprises about 400IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, thesecond expansion culture media comprises about 300 IU/mL of IL-15 toabout 100 IU/mL of IL-15. In some embodiments, the second expansionculture media comprises about 200 IU/mL of IL-15. In some embodiments,the cell culture medium comprises about 180 IU/mL of IL-15. In anembodiment, the cell culture medium further comprises IL-15. In apreferred embodiment, the cell culture medium comprises about 180 IU/mLof IL-15.

In some embodiments, the second expansion culture media comprises about20 IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21,about 10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21,about 3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21,or about 0.5 IU/mL of IL-21. In some embodiments, the second expansionculture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL ofIL-21. In some embodiments, the second expansion culture media comprisesabout 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In someembodiments, the second expansion culture media comprises about 12 IU/mLof IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the secondexpansion culture media comprises about 10 IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the second expansion culture mediacomprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In someembodiments, the second expansion culture media comprises about 2 IU/mLof IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL of IL-21. In some embodiments, the cell culture medium comprisesabout 0.5 IU/mL of IL-21. In an embodiment, the cell culture mediumfurther comprises IL-21. In a preferred embodiment, the cell culturemedium comprises about 1 IU/mL of IL-21.

In some embodiments the antigen-presenting feeder cells (APCs) arePBMCs. In an embodiment, the ratio of TILs to PBMCs and/orantigen-presenting cells in the rapid expansion and/or the secondexpansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225,about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In anembodiment, the ratio of TILs to PBMCs in the rapid expansion and/or thesecond expansion is between 1 to 50 and 1 to 300. In an embodiment, theratio of TILs to PBMCs in the rapid expansion and/or the secondexpansion is between 1 to 100 and 1 to 200.

In an embodiment, REP and/or the second expansion is performed in flaskswith the bulk TILs being mixed with a 100- or 200-fold excess ofinactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody and 3000 IU/mLIL-2 in 150 ml media. Media replacement is done (generally ⅔ mediareplacement via respiration with fresh media) until the cells aretransferred to an alternative growth chamber. Alternative growthchambers include G-REX flasks and gas permeable containers as more fullydiscussed below.

In some embodiments, the second expansion (which can include processesreferred to as the REP process) is shortened to 7-14 days, as discussedin the examples and figures. In some embodiments, the second expansionis shortened to 11 days.

In an embodiment, REP and/or the second expansion may be performed usingT-175 flasks and gas permeable bags as previously described (Tran, etal., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother.2003, 26, 332-42) or gas permeable cultureware (G-Rex flasks). In someembodiments, the second expansion (including expansions referred to asrapid expansions) is performed in T-175 flasks, and about 1×10⁶ TILssuspended in 150 mL of media may be added to each T-175 flask. The TILsmay be cultured in a 1 to 1 mixture of CM and AIM-V medium, supplementedwith 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3. The T-175flasks may be incubated at 37° C. in 5% CO2. Half the media may beexchanged on day 5 using 50/50 medium with 3000 IU per mL of IL-2. Insome embodiments, on day 7 cells from two T-175 flasks may be combinedin a 3 L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU permL of IL-2 was added to the 300 ml of TIL suspension. The number ofcells in each bag was counted every day or two and fresh media was addedto keep the cell count between 0.5 and 2.0×10⁶ cells/mL.

In an embodiment, the second expansion (which can include expansionsreferred to as REP, as well as those referred to in Step D of FIG. 9 )may be performed in 500 mL capacity gas permeable flasks with 100 cmgas-permeable silicon bottoms (G-Rex 100, commercially available fromWilson Wolf Manufacturing Corporation, New Brighton, Minn., USA), 5×10⁶or 10×10⁶ TIL may be cultured with PBMCs in 400 mL of 50/50 medium,supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ngper ml of anti-CD3 (OKT3). The G-Rex 100 flasks may be incubated at 37°C. in 5% CO2. On day 5, 250 mL of supernatant may be removed and placedinto centrifuge bottles and centrifuged at 1500 rpm (491×g) for 10minutes. The TIL pellets may be re-suspended with 150 mL of fresh mediumwith 5% human AB serum, 3000 IU per mL of IL-2, and added back to theoriginal G-Rex 100 flasks. When TIL are expanded serially in G-Rex 100flasks, on day 7 the TIL in each G-Rex 100 may be suspended in the 300mL of media present in each flask and the cell suspension may be dividedinto 3 100 mL aliquots that may be used to seed 3 G-Rex 100 flasks. Then150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may beadded to each flask. The G-Rex 100 flasks may be incubated at 37° C. in5% CO2 and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 maybe added to each G-REX 100 flask. The cells may be harvested on day 14of culture.

In an embodiment, the second expansion (including expansions referred toas REP) is performed in flasks with the bulk TILs being mixed with a100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3anti-CD3 antibody and 3000 IU/mL IL-2 in 150 ml media. In someembodiments, media replacement is done until the cells are transferredto an alternative growth chamber. In some embodiments, ⅔ of the media isreplaced by respiration with fresh media. In some embodiments,alternative growth chambers include G-REX flasks and gas permeablecontainers as more fully discussed below.

In an embodiment, the second expansion (including expansions referred toas REP) is performed and further comprises a step wherein TILs areselected for superior tumor reactivity. Any selection method known inthe art may be used. For example, the methods described in U.S. PatentApplication Publication No. 2016/0010058 A1, the disclosures of whichare incorporated herein by reference, may be used for selection of TILsfor superior tumor reactivity.

Optionally, a cell viability assay can be performed after the secondexpansion (including expansions referred to as the REP expansion), usingstandard assays known in the art. For example, a trypan blue exclusionassay can be done on a sample of the bulk TILs, which selectively labelsdead cells and allows a viability assessment. In some embodiments, TILsamples can be counted and viability determined using a Cellometer K2automated cell counter (Nexcelom Bioscience, Lawrence, Mass.). In someembodiments, viability is determined according to the Cellometer K2Image Cytometer Automatic Cell Counter protocol described, for example,in Example 15.

In some embodiments, the second expansion (including expansions referredto as REP) of TIL can be performed using T-175 flasks and gas-permeablebags as previously described (Tran K Q, Zhou J, Durflinger K H, et al.,2008, J Immunother, 31:742-751, and Dudley M E, Wunderlich J R, SheltonT E, et al. 2003, J Immunother., 26:332-342) or gas-permeable G-Rexflasks. In some embodiments, the second expansion is performed usingflasks. In some embodiments, the second expansion is performed usinggas-permeable G-Rex flasks. In some embodiments, the second expansion isperformed in T-175 flasks, and about 1×10⁶ TIL are suspended in about150 mL of media and this is added to each T-175 flask. The TIL arecultured with irradiated (50 Gy) allogeneic PBMC as “feeder” cells at aratio of 1 to 100 and the cells were cultured in a 1 to 1 mixture of CMand AIM-V medium (50/50 medium), supplemented with 3000 IU/mL of IL-2and 30 ng/mL of anti-CD3. The T-175 flasks are incubated at 37° C. in 5%CO2. In some embodiments, half the media is changed on day 5 using 50/50medium with 3000 IU/mL of IL-2. In some embodiments, on day 7, cellsfrom 2 T-175 flasks are combined in a 3 L bag and 300 mL of AIM-V with5% human AB serum and 3000 IU/mL of IL-2 is added to the 300 mL of TILsuspension. The number of cells in each bag can be counted every day ortwo and fresh media can be added to keep the cell count between about0.5 and about 2.0×10⁶ cells/mL.

In some embodiments, the second expansion (including expansions referredto as REP) are performed in 500 mL capacity flasks with 100 cm²gas-permeable silicon bottoms (G-Rex 100, Wilson Wolf) (FIG. 1 ), about5×10⁶ or 10×10⁶ TIL are cultured with irradiated allogeneic PBMC at aratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 3000IU/mL of IL-2 and 30 ng/mL of anti-CD3. The G-Rex 100 flasks areincubated at 37° C. in 5% CO₂. In some embodiments, on day 5, 250 mL ofsupernatant is removed and placed into centrifuge bottles andcentrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellets can thenbe resuspended with 150 mL of fresh 50/50 medium with 3000 IU/mL of IL-2and added back to the original G-Rex 100 flasks. In embodiments whereTILs are expanded serially in G-Rex 100 flasks, on day 7 the TIL in eachG-Rex 100 are suspended in the 300 mL of media present in each flask andthe cell suspension was divided into three 100 mL aliquots that are usedto seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serumand 3000 IU/mL of IL-2 is added to each flask. The G-Rex 100 flasks areincubated at 37° C. in 5% CO₂ and after 4 days 150 mL of AIM-V with 3000IU/mL of IL-2 is added to each G-Rex 100 flask. The cells are harvestedon day 14 of culture.

The diverse antigen receptors of T and B lymphocytes are produced bysomatic recombination of a limited, but large number of gene segments.These gene segments: V (variable), D (diversity), J (joining), and C(constant), determine the binding specificity and downstreamapplications of immunoglobulins and T-cell receptors (TCRs). The presentinvention provides a method for generating TILs which exhibit andincrease the T-cell repertoire diversity. In some embodiments, the TILsobtained by the present method exhibit an increase in the T-cellrepertoire diversity. In some embodiments, the TILs obtained in thesecond expansion exhibit an increase in the T-cell repertoire diversity.In some embodiments, the increase in diversity is an increase in theimmunoglobulin diversity and/or the T-cell receptor diversity. In someembodiments, the diversity is in the immunoglobulin is in theimmunoglobulin heavy chain. In some embodiments, the diversity is in theimmunoglobulin is in the immunoglobulin light chain. In someembodiments, the diversity is in the T-cell receptor. In someembodiments, the diversity is in one of the T-cell receptors selectedfrom the group consisting of alpha, beta, gamma, and delta receptors. Insome embodiments, there is an increase in the expression of T-cellreceptor (TCR) alpha and/or beta. In some embodiments, there is anincrease in the expression of T-cell receptor (TCR) alpha. In someembodiments, there is an increase in the expression of T-cell receptor(TCR) beta. In some embodiments, there is an increase in the expressionof TCRab (i.e., TCRα/β).

In some embodiments, the second expansion culture medium (e.g.,sometimes referred to as CM2 or the second cell culture medium),comprises IL-2, OKT-3, as well as the antigen-presenting feeder cells(APCs), as discussed in more detail below.

In some embodiments, the second expansion, for example, Step D accordingto FIG. 9 , is performed in a closed system bioreactor. In someembodiments, a closed system is employed for the TIL expansion, asdescribed herein. In some embodiments, a single bioreactor is employed.In some embodiments, the single bioreactor employed is for example aG-REX-10 or a G-REX-100. In some embodiments, the closed systembioreactor is a single bioreactor.

1. Feeder Cells and Antigen Presenting Cells

In an embodiment, the second expansion procedures described herein (forexample including expansion such as those described in Step D from FIG.9 , as well as those referred to as REP) require an excess of feedercells during REP TIL expansion and/or during the second expansion. Inmany embodiments, the feeder cells are peripheral blood mononuclearcells (PBMCs) obtained from standard whole blood units from healthyblood donors. The PBMCs are obtained using standard methods such asFicoll-Paque gradient separation.

In general, the allogenic PBMCs are inactivated, either via irradiationor heat treatment, and used in the REP procedures, as described in theexamples, in particular example 14, which provides an exemplary protocolfor evaluating the replication incompetence of irradiate allogeneicPBMCs.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells on day 14 is less than the initial viablecell number put into culture on day 0 of the REP and/or day 0 of thesecond expansion (i.e., the start day of the second expansion). See, forexample, Example 14.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells, cultured in the presence of OKT3 and IL-2,on day 7 and day 14 has not increased from the initial viable cellnumber put into culture on day 0 of the REP and/or day 0 of the secondexpansion (i.e., the start day of the second expansion). In someembodiments, the PBMCs are cultured in the presence of 30 ng/ml OKT3antibody and 3000 IU/ml IL-2. See, for example, Example 13.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells, cultured in the presence of OKT3 and IL-2,on day 7 and day 14 has not increased from the initial viable cellnumber put into culture on day 0 of the REP and/or day 0 of the secondexpansion (i.e., the start day of the second expansion). In someembodiments, the PBMCs are cultured in the presence of 5-60 ng/ml OKT3antibody and 1000-6000 IU/ml IL-2. In some embodiments, the PBMCs arecultured in the presence of 10-50 ng/ml OKT3 antibody and 2000-5000IU/ml IL-2. In some embodiments, the PBMCs are cultured in the presenceof 20-40 ng/ml OKT3 antibody and 2000-4000 IU/ml IL-2. In someembodiments, the PBMCs are cultured in the presence of 25-35 ng/ml OKT3antibody and 2500-3500 IU/ml IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. Insome embodiments, the antigen-presenting feeder cells are artificialantigen-presenting feeder cells. In an embodiment, the ratio of TILs toantigen-presenting feeder cells in the second expansion is about 1 to25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375,about 1 to 400, or about 1 to 500. In an embodiment, the ratio of TILsto antigen-presenting feeder cells in the second expansion is between 1to 50 and 1 to 300. In an embodiment, the ratio of TILs toantigen-presenting feeder cells in the second expansion is between 1 to100 and 1 to 200.

In an embodiment, the second expansion procedures described hereinrequire a ratio of about 2.5×10⁹ feeder cells to about 100×10⁶ TILs. Inanother embodiment, the second expansion procedures described hereinrequire a ratio of about 2.5×10⁹ feeder cells to about 50×10⁶ TILs. Inyet another embodiment, the second expansion procedures described hereinrequire about 2.5×10⁹ feeder cells to about 25×10⁶ TILs.

In an embodiment, the second expansion procedures described hereinrequire an excess of feeder cells during the second expansion. In manyembodiments, the feeder cells are peripheral blood mononuclear cells(PBMCs) obtained from standard whole blood units from healthy blooddonors. The PBMCs are obtained using standard methods such asFicoll-Paque gradient separation. In an embodiment, artificialantigen-presenting (aAPC) cells are used in place of PBMCs.

In general, the allogenic PBMCs are inactivated, either via irradiationor heat treatment, and used in the TIL expansion procedures describedherein, including the exemplary procedures as described in FIGS. 5, 6,8, 9, 10, and 11 .

In an embodiment, artificial antigen presenting cells are used in thesecond expansion as a replacement for, or in combination with, PBMCs.

2. Cytokines

The expansion methods described herein generally use culture media withhigh doses of a cytokine, in particular IL-2, as is known in the art.

Alternatively, using combinations of cytokines for the rapid expansionand or second expansion of TILS is additionally possible, withcombinations of two or more of IL-2, IL-15 and IL-21 as is generallyoutlined in International Publication No. WO 2015/189356 and WInternational Publication No. WO 2015/189357, hereby expresslyincorporated by reference in their entirety. Thus, possible combinationsinclude IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, IL-15and IL-21, with the latter finding particular use in many embodiments.The use of combinations of cytokines specifically favors the generationof lymphocytes, and in particular T-cells as described therein.

3. Anti-CD3 Antibodies

In some embodiments, the culture media used in expansion methodsdescribed herein (including those referred to as REP, see for example,FIG. 9 ) also includes an anti-CD3 antibody. An anti-CD3 antibody incombination with IL-2 induces T-cell activation and cell division in theTIL population. This effect can be seen with full length antibodies aswell as Fab and F(ab′)2 fragments, with the former being generallypreferred; see, e.g., Tsoukas et al., J. Immunol. 1985, 135, 1719,hereby incorporated by reference in its entirety.

As will be appreciated by those in the art, there are a number ofsuitable anti-human CD3 antibodies that find use in the invention,including anti-human CD3 polyclonal and monoclonal antibodies fromvarious mammals, including, but not limited to, murine, human, primate,rat, and canine antibodies. In particular embodiments, the OKT3 anti-CD3antibody is used (commercially available from Ortho-McNeil, Raritan,N.J. or Miltenyi Biotech, Auburn, Calif.).

E. STEP E: Harvest TILS

After the second expansion step, cells can be harvested. In someembodiments the TILs are harvested after one, two, three, four or moreexpansion steps, for example as provided in FIG. 9 . In some embodimentsthe TILs are harvested after two expansion steps, for example asprovided in FIG. 9 .

TILs can be harvested in any appropriate and sterile manner, includingfor example by centrifugation. Methods for TIL harvesting are well knownin the art and any such know methods can be employed with the presentprocess. In some embodiments, TILS are harvest using an automatedsystem.

Cell harvesters and/or cell processing systems are commerciallyavailable from a variety of sources, including, for example, FreseniusKabi, Tomtec Life Science, Perkin Elmer, and Inotech BiosystemsInternational, Inc. Any cell based harvester can be employed with thepresent methods. In some embodiments, the cell harvester and/or cellprocessing systems is a membrane-based cell harvester. In someembodiments, cell harvesting is via a cell processing system, such asthe LOVO system (manufactured by Fresenius Kabi). The term “LOVO cellprocessing system” also refers to any instrument or device manufacturedby any vendor that can pump a solution comprising cells through amembrane or filter such as a spinning membrane or spinning filter in asterile and/or closed system environment, allowing for continuous flowand cell processing to remove supernatant or cell culture media withoutpelletization. In some embodiments, the cell harvester and/or cellprocessing system can perform cell separation, washing, fluid-exchange,concentration, and/or other cell processing steps in a closed, sterilesystem.

In some embodiments, the harvest, for example, Step E according to FIG.9 , is performed from a closed system bioreactor. In some embodiments, aclosed system is employed for the TIL expansion, as described herein. Insome embodiments, a single bioreactor is employed. In some embodiments,the single bioreactor employed is for example a G-REX-10 or a G-REX-100.In some embodiments, the closed system bioreactor is a singlebioreactor.

F. STEP F: Final Formulation/Transfer to Infusion Bag

After Steps A through E as provided in an exemplary order in FIG. 9 andas outlined in detailed above and herein are complete, cells aretransferred to a container for use in administration to a patient. Insome embodiments, once a therapeutically sufficient number of TILs areobtained using the expansion methods described above, they aretransferred to a container for use in administration to a patient.

In an embodiment, TILs expanded using APCs of the present disclosure areadministered to a patient as a pharmaceutical composition. In anembodiment, the pharmaceutical composition is a suspension of TILs in asterile buffer. TILs expanded using PBMCs of the present disclosure maybe administered by any suitable route as known in the art. In someembodiments, the T-cells are administered as a single intra-arterial orintravenous infusion, which preferably lasts approximately 30 to 60minutes. Other suitable routes of administration includeintraperitoneal, intrathecal, and intralymphatic.

1. Pharmaceutical Compositions, Dosages, and Dosing Regimens

In an embodiment, TILs expanded using the methods of the presentdisclosure are administered to a patient as a pharmaceuticalcomposition. In an embodiment, the pharmaceutical composition is asuspension of TILs in a sterile buffer. TILs expanded using PBMCs of thepresent disclosure may be administered by any suitable route as known inthe art. In some embodiments, the T-cells are administered as a singleintra-arterial or intravenous infusion, which preferably lastsapproximately 30 to 60 minutes. Other suitable routes of administrationinclude intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TILs can be administered. In some embodiments, fromabout 2.3×10¹⁰ to about 13.7×10¹⁰ TILs are administered, with an averageof around 7.8×10¹⁰ TILs, particularly if the cancer is melanoma. In anembodiment, about 1.2×10¹⁰ to about 4.3×10¹⁰ of TILs are administered.In some embodiments, about 3×10¹⁰ to about 12×10¹⁰ TILs areadministered. In some embodiments, about 4×10¹⁰ to about 10×10¹⁰ TILsare administered. In some embodiments, about 5×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, about 6×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, about 7×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, the therapeutically effectivedosage is about 2.3×10¹⁰ to about 13.7×10¹⁰. In some embodiments, thetherapeutically effective dosage is about 7.8×10¹⁰ TILs, particularly ofthe cancer is melanoma. In some embodiments, the therapeuticallyeffective dosage is about 1.2×10¹⁰ to about 4.3×10¹⁰ of TILs. In someembodiments, the therapeutically effective dosage is about 3×10¹⁰ toabout 12×10¹⁰ TILs. In some embodiments, the therapeutically effectivedosage is about 4×10¹⁰ to about 10×10¹⁰ TILs. In some embodiments, thetherapeutically effective dosage is about 5×10¹⁰ to about 8×10¹⁰ TILs.In some embodiments, the therapeutically effective dosage is about6×10¹⁰ to about 8×10¹⁰ TILs. In some embodiments, the therapeuticallyeffective dosage is about 7×10¹⁰ to about 8×10¹⁰ TILs.

In some embodiments, the number of the TILs provided in thepharmaceutical compositions of the invention is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰ 2×10¹⁰, 3×10¹⁰, 4×10¹⁰,5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹,5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹¹, 4×10¹¹,5×10¹², 6×10¹¹, 7×10¹², 8×10¹¹, 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹¹,5×10¹³, 6×10¹¹, 6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In an embodiment,the number of the TILs provided in the pharmaceutical compositions ofthe invention is in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷to 5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to5×10⁹, 5×10⁹ to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to1×10¹², 1×10¹² to 5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is less than, for example,100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%,0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%,0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceuticalcomposition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is greater than 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%,18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25%16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%,13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25%11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%,8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%,5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%,2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%,0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001%w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% toabout 27%, about 0.05% to about 26%, about 0.06% to about 25%, about0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%,about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% toabout 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9%to about 12% or about 1% to about 10% w/w, w/v or v/v of thepharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%,about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% toabout 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/vor v/v of the pharmaceutical composition.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is equal to or less than 10g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g,4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g,0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g,0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or0.0001 g.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is more than 0.0001 g,0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g,0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g,0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g,0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g,0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or10 g.

The TILs provided in the pharmaceutical compositions of the inventionare effective over a wide dosage range. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the gender and age of the subject to be treated, the bodyweight of the subject to be treated, and the preference and experienceof the attending physician. The clinically-established dosages of theTILs may also be used if appropriate. The amounts of the pharmaceuticalcompositions administered using the methods herein, such as the dosagesof TILs, will be dependent on the human or mammal being treated, theseverity of the disorder or condition, the rate of administration, thedisposition of the active pharmaceutical ingredients and the discretionof the prescribing physician.

In some embodiments, TILs may be administered in a single dose. Suchadministration may be by injection, e.g., intravenous injection. In someembodiments, TILs may be administered in multiple doses. Dosing may beonce, twice, three times, four times, five times, six times, or morethan six times per year. Dosing may be once a month, once every twoweeks, once a week, or once every other day. Administration of TILs maycontinue as long as necessary.

In some embodiments, an effective dosage of TILs is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹°, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹²,6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In some embodiments, an effectivedosage of TILs is in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷to 5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹ 1×10⁹ to 5×10⁹,5×10⁹ to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to 1×10¹²,1×10¹² to 5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, an effective dosage of TILs is in the range ofabout 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg toabout 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kgto about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kgto about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kgto about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg toabout 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kgmg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dosage of TILs is in the range ofabout 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg toabout 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg,about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg toabout 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg,about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg toabout 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg,or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg,about 195 mg to about 205 mg, or about 198 to about 207 mg.

An effective amount of the TILs may be administered in either single ormultiple doses by any of the accepted modes of administration of agentshaving similar utilities, including intranasal and transdermal routes,by intra-arterial injection, intravenously, intraperitoneally,parenterally, intramuscularly, subcutaneously, topically, bytransplantation, or by inhalation.

G. Optional Cell Viability Analyses

Optionally, a cell viability assay can be performed after the Step Bfirst expansion, using standard assays known in the art. For example, atrypan blue exclusion assay can be done on a sample of the bulk TILs,which selectively labels dead cells and allows a viability assessment.Other assays for use in testing viability can include but are notlimited to the Alamar blue assay; and the MTT assay.

1. Cell Counts, Viability, Flow Cytometry

In some embodiments, cell counts and/or viability are measured. Theexpression of markers such as but not limited CD3, CD4, CD8, and CD56,as well as any other disclosed or described herein, can be measured byflow cytometry with antibodies, for example but not limited to thosecommercially available from BD Bio-sciences (BD Biosciences, San Jose,Calif.) using a FACSCanto™ flow cytometer (BD Biosciences). The cellscan be counted manually using a disposable c-chip hemocytometer (VWR,Batavia, Ill.) and viability can be assessed using any method known inthe art, including but not limited to trypan blue staining.

In some cases, the bulk TIL population can be cryopreserved immediately,using the protocols discussed below. Alternatively, the bulk TILpopulation can be subjected to REP and then cryopreserved as discussedbelow. Similarly, in the case where genetically modified TILs will beused in therapy, the bulk or REP TIL populations can be subjected togenetic modifications for suitable treatments.

2. Cell Cultures

In an embodiment, a method for expanding TILs may include using about5,000 mL to about 25,000 mL of cell medium, about 5,000 mL to about10,000 mL of cell medium, or about 5,800 mL to about 8,700 mL of cellmedium. In an embodiment, expanding the number of TILs uses no more thanone type of cell culture medium. Any suitable cell culture medium may beused, e.g., AIM-V cell medium (L-glutamine, 50 μM streptomycin sulfate,and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, CarlsbadCalif.). In this regard, the inventive methods advantageously reduce theamount of medium and the number of types of medium required to expandthe number of TIL. In an embodiment, expanding the number of TIL maycomprise adding fresh cell culture media to the cells (also referred toas feeding the cells) no more frequently than every third or fourth day.Expanding the number of cells in a gas permeable container simplifiesthe procedures necessary to expand the number of cells by reducing thefeeding frequency necessary to expand the cells.

In an embodiment, the cell medium in the first and/or second gaspermeable container is unfiltered. The use of unfiltered cell medium maysimplify the procedures necessary to expand the number of cells. In anembodiment, the cell medium in the first and/or second gas permeablecontainer lacks beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TILs from the tumor tissue sample; expanding the number ofTILs in a second gas permeable container containing cell medium thereinusing aAPCs for a duration of about 14 to about 42 days, e.g., about 28days.

In an embodiment, TILs are expanded in gas-permeable containers.Gas-permeable containers have been used to expand TILs using PBMCs usingmethods, compositions, and devices known in the art, including thosedescribed in U.S. Patent Application Publication No. 2005/0106717 A1,the disclosures of which are incorporated herein by reference. In anembodiment, TILs are expanded in gas-permeable bags. In an embodiment,TILs are expanded using a cell expansion system that expands TILs in gaspermeable bags, such as the Xuri Cell Expansion System W25 (GEHealthcare). In an embodiment, TILs are expanded using a cell expansionsystem that expands TILs in gas permeable bags, such as the WAVEBioreactor System, also known as the Xuri Cell Expansion System W5 (GEHealthcare). In an embodiment, the cell expansion system includes a gaspermeable cell bag with a volume selected from the group consisting ofabout 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL,about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L,about 9 L, and about 10 L. In an embodiment, TILs can be expanded inG-Rex flasks (commercially available from Wilson Wolf Manufacturing).Such embodiments allow for cell populations to expand from about 5×10⁵cells/cm² to between 10×10⁶ and 30×10⁶ cells/cm². In an embodiment thisexpansion is conducted without adding fresh cell culture media to thecells (also referred to as feeding the cells). In an embodiment, this iswithout feeding so long as medium resides at a height of about 10 cm inthe G-Rex flask. In an embodiment this is without feeding but with theaddition of one or more cytokines. In an embodiment, the cytokine can beadded as a bolus without any need to mix the cytokine with the medium.Such containers, devices, and methods are known in the art and have beenused to expand TILs, and include those described in U.S. PatentApplication Publication No. US 2014/0377739A1, International PublicationNo. WO 2014/210036 A1, U.S. Patent Application Publication No. us2013/0115617 A1, International Publication No. WO 2013/188427 A1, U.S.Patent Application Publication No. US 2011/0136228 A1, U.S. Pat. No.8,809,050 B2, International publication No. WO 2011/072088 A2, U.S.Patent Application Publication No. US 2016/0208216 A1, U.S. PatentApplication Publication No. US 2012/0244133 A1, InternationalPublication No. WO 2012/129201 A1, U.S. Patent Application PublicationNo. US 2013/0102075 A1, U.S. Pat. No. 8,956,860 B2, InternationalPublication No. WO 2013/173835 A1, U.S. Patent Application PublicationNo. US 2015/0175966 A1, the disclosures of which are incorporated hereinby reference. Such processes are also described in Jin et al., J.Immunotherapy, 2012, 35:283-292. Optional Genetic Engineering of TILs

In some embodiments, the TILs are optionally genetically engineered toinclude additional functionalities, including, but not limited to, ahigh-affinity T-cell receptor (TCR), e.g., a TCR targeted at atumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or achimeric antigen receptor (CAR) which binds to a tumor-associated cellsurface molecule (e.g., mesothelin) or lineage-restricted cell surfacemolecule (e.g., CD19).

H. Optional Cryopreservation of TILs

As discussed above, and exemplified in Steps A through E as provided inFIG. 9 , cryopreservation can occur at numerous points throughout theTIL expansion process. In some embodiments, the expanded population ofTILs after the second expansion (as provided for example, according toStep D of FIG. 9 ) can be cryopreserved. Cryopreservation can begenerally accomplished by placing the TIL population into a freezingsolution, e.g., 85% complement inactivated AB serum and 15% dimethylsulfoxide (DMSO). The cells in solution are placed into cryogenic vialsand stored for 24 hours at −80° C., with optional transfer to gaseousnitrogen freezers for cryopreservation. See Sadeghi, et al., ActaOncologica 2013, 52, 978-986. In some embodiments, the TILs arecryopreserved in 5% DMSO. In some embodiments, the TILs arecryopreserved in cell culture media plus 5% DMSO. In some embodiments,the TILs are cryopreserved according to the methods provided in Examples8 and 9.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately ⅘ of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

I. Phenotypic Characteristics of Expanded TILs

In some embodiment, the TILs are analyzed for expression of numerousphenotype markers after expansion, including those described herein andin the Examples. In an embodiment, expression of one or more phenotypicmarkers is examined. In some embodiments, the phenotypic characteristicsof the TILs are analyzed after the first expansion in Step B. In someembodiments, the phenotypic characteristics of the TILs are analyzedduring the transition in Step C. In some embodiments, the phenotypiccharacteristics of the TILs are analyzed during the transition accordingto Step C and after cryopreservation. In some embodiments, thephenotypic characteristics of the TILs are analyzed after the secondexpansion according to Step D. In some embodiments, the phenotypiccharacteristics of the TILs are analyzed after two or more expansionsaccording to Step D. In some embodiments, the marker is selected fromthe group consisting of TCRab (i.e., TCRα/β), CD57, CD28, CD4, CD27,CD56, CD8a, CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In someembodiments, the marker is selected from the group consisting of TCRab(i.e., TCRα/β), CD57, CD28, CD4, CD27, CD56, and CD8a. In an embodiment,the marker is selected from the group consisting of CD45RA, CD8a, CCR7,CD4, CD3, CD38, and HLA-DR. In some embodiments, expression of one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, or fourteen markers is examined. In some embodiments, theexpression from one or more markers from each group is examined. In someembodiments, one or more of HLA-DR, CD38, and CD69 expression ismaintained (i.e., does not exhibit a statistically significantdifference) in fresh TILs as compared to thawed TILs. In someembodiments, the activation status of TILs is maintained in the thawedTILs.

In an embodiment, expression of one or more regulatory markers ismeasured. In some embodiments, the regulatory marker is selected fromthe group consisting of CD137, CD8a, Lag3, CD4, CD3, PD-1, TIM-3, CD69,CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, theregulatory marker is selected from the group consisting of CD137, CD8a,Lag3, CD4, CD3, PD-1, and TIM-3. In some embodiments, the regulatorymarker is selected from the group consisting of CD69, CD8a, TIGIT, CD4,CD3, KLRG1, and CD154. In some embodiments, regulatory moleculeexpression is decreased in thawed TILs as compared to fresh TILs. Insome embodiments, expression of regulatory molecules LAG-3 and TIM-3 isdecreased in thawed TILs as compared to fresh TILs. In some embodiments,there is no significant difference in CD4, CD8, NK, TCRαβ expression. Insome embodiments, there is no significant difference in CD4, CD8, NK,TCRαβ expression, and/or memory markers in fresh TILs as compared tothawed TILs. In some embodiments, there is no significant difference inCD4, CD8, NK, TCRαβ expression between the TILs produced by the methodsprovided herein, as exemplified for example in FIG. 9 , and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 .

In some embodiments, no selection of the first population of TILs,second population of TILs, third population of TILs, harvested TILpopulation, and/or the therapeutic TIL population based on CD4, CD8,and/or NK, TCRαβ expression is performed during any of steps, includingthose discussed above or as provided for example in FIG. 9 . In someembodiments, no selection of the first population of TILs based on CD4,CD8, and/or NK, TCRαβ is performed. In some embodiments, no selection ofthe second population of TILs based on CD4, CD8, and/or NK, TCRαβexpression is performed. In some embodiments, no selection of the thirdpopulation of TILs based on CD4, CD8, and/or NK, TCRαβ expression isperformed. In some embodiments, no selection of the harvested populationof TILs based on CD4, CD8, and/or NK, TCRαβ expression is performed. Insome embodiments, no selection of the therapeutic population of TILsbased on CD4, CD8, and/or NK, TCRαβ expression is performed.

In an embodiment, no selection of the first population of TILs, secondpopulation of TILs, third population of TILs, or harvested TILpopulation based on CD4, CD8, and/or NK, TCRαβ expression is performedduring any of steps (a) to (f) of the method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILscomprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T-cells and/or central memory T-cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system.

In an embodiment, no selection of the first population of TILs, secondpopulation of TILs, third population of TILs, or harvested TILpopulation based on CD4, CD8, and/or NK, TCRαβ expression is performedduring any of steps (a) to (h) of the method for treating a subject withcancer, the method comprising administering expanded tumor infiltratinglymphocytes (TILs) comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a subject by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T-cells and/or central memory T-cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system;    -   (g) optionally cryopreserving the infusion bag comprising the        harvested TIL population from step (f) using a cryopreservation        process; and    -   (h) administering a therapeutically effective dosage of the        third population of TILs from the infusion bag in step (g) to        the patient.

In some embodiments the memory marker is selected from the groupconsisting of CCR7 and CD62L

In some embodiments, the viability of the fresh TILs as compared to thethawed TILs is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 98%. In some embodiments, theviability of both the fresh and thawed TILs is greater than 70%, greaterthan 75%, greater than 80%, greater than 85%, greater than 90%, greaterthan 95%, or greater than 98%. In some embodiments, the viability ofboth the fresh and thawed product is greater than 80%, greater than 81%,greater than 82%, greater than 83%, greater than 84%, greater than 85%,greater than 86%, greater than 87%, greater than 88%, greater than 89%,or greater than 90%. In some embodiments, the viability of both thefresh and thawed product is greater than 86%.

In an embodiment, restimulated TILs can also be evaluated for cytokinerelease, using cytokine release assays. In some embodiments, TILs can beevaluated for interferon-7 (IFN-7) secretion in response to stimulationeither with OKT3 or co-culture with autologous tumor digest. Forexample, in embodiments employing OKT3 stimulation, TILs are washedextensively, and duplicate wells are prepared with 1×10⁵ cells in 0.2 mLCM in 96-well flat-bottom plates precoated with 0.1 or 1.0 μg/mL of OKT3diluted in phosphate-buffered saline. After overnight incubation, thesupernatants are harvested and IFN-7 in the supernatant is measured byELISA (Pierce/Endogen, Woburn, Mass.). For the co-culture assay, 1×10⁵TIL cells are placed into a 96-well plate with autologous tumor cells.(1:1 ratio). After a 24-hour incubation, supernatants are harvested andIFN-7 release can be quantified, for example by ELISA.

Flow cytometric analysis of cell surface biomarkers: TIL samples werealiquoted for flow cytometric analysis of cell surface markers see, forExample see Examples 7, 8, and 9.

In some embodiments, the TILs are being evaluated for various regulatorymarkers. In some embodiments, the regulatory marker is selected from thegroup consisting of TCR α/β, CD56, CD27, CD28, CD57, CD45RA, CD45RO,CD25, CD127, CD95, IL-2R-, CCR7, CD62L, KLRG1, and CD122. In someembodiments, the regulatory marker is TCR α/β. In some embodiments, theregulatory marker is CD56. In some embodiments, the regulatory marker isCD27. In some embodiments, the regulatory marker is CD28. In someembodiments, the regulatory marker is CD57. In some embodiments, theregulatory marker is CD45RA. In some embodiments, the regulatory markeris CD45RO. In some embodiments, the regulatory marker is CD25. In someembodiments, the regulatory marker is CD127. In some embodiments, theregulatory marker is CD95. In some embodiments, the regulatory marker isIL-2R-. In some embodiments, the regulatory marker is CCR7. In someembodiments, the regulatory marker is CD62L. In some embodiments, theregulatory marker is KLRG1. In some embodiments, the regulatory markeris CD122.

In an embodiment, the expanded TILs are analyzed for expression ofnumerous phenotype markers, including those described herein and in theExamples. In an embodiment, expression of one or more phenotypic markersis examined. In some embodiments, the marker is selected from the groupconsisting of TCRab (i.e., TCRα/β), CD57, CD28, CD4, CD27, CD56, CD8a,CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, themarker is selected from the group consisting of TCRab (i.e., TCRα/β),CD57, CD28, CD4, CD27, CD56, and CD8a. In an embodiment, the marker isselected from the group consisting of CD45RA, CD8a, CCR7, CD4, CD3,CD38, and HLA-DR. In some embodiments, expression of one, two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, orfourteen markers is examined. In some embodiments, the expression fromone or more markers from each group is examined. In some embodiments,one or more of HLA-DR, CD38, and CD69 expression is maintained (i.e.,does not exhibit a statistically significant difference) in fresh TILsas compared to thawed TILs. In some embodiments, the activation statusof TILs is maintained in the thawed TILs.

In an embodiment, expression of one or more regulatory markers ismeasured. In some embodiments, the regulatory marker is selected fromthe group consisting of CD137, CD8a, Lag3, CD4, CD3, PD1, TIM-3, CD69,CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, theregulatory marker is selected from the group consisting of CD137, CD8a,Lag3, CD4, CD3, PD1, and TIM-3. In some embodiments, the regulatorymarker is selected from the group consisting of CD69, CD8a, TIGIT, CD4,CD3, KLRG1, and CD154. In some embodiments, regulatory moleculeexpression is decreased in thawed TILs as compared to fresh TILs. Insome embodiments, expression of regulatory molecules LAG-3 and TIM-3 isdecreased in thawed TILs as compared to fresh TILs. In some embodiments,there is no significant difference in CD4, CD8, NK, TCRαβ expression. Insome embodiments, there is no significant difference in CD4, CD8, NK,TCRαβ expression, and/or memory markers in fresh TILs as compared tothawed TILs.

In some embodiments the memory marker is selected from the groupconsisting of CCR7 and CD62L.

In some embodiments, the viability of the fresh TILs as compared to thethawed TILs is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 98%. In some embodiments, theviability of both the fresh and thawed TILs is greater than 70%, greaterthan 75%, greater than 80%, greater than 85%, greater than 90%, greaterthan 95%, or greater than 98%. In some embodiments, the viability ofboth the fresh and thawed product is greater than 80%, greater than 81%,greater than 82%, greater than 83%, greater than 84%, greater than 85%,greater than 86%, greater than 87%, greater than 88%, greater than 89%,or greater than 90%. In some embodiments, the viability of both thefresh and thawed product is greater than 86%.

In an embodiment, restimulated TILs can also be evaluated for cytokinerelease, using cytokine release assays. In some embodiments, TILs can beevaluated for interferon-7 (IFN-7) secretion in response to stimulationeither with OKT3 or coculture with autologous tumor digest. For example,in embodiments employing OKT3 stimulation, TILs are washed extensively,and duplicate wells are prepared with 1×10⁵ cells in 0.2 mL CM in96-well flat-bottom plates precoated with 0.1 or 1.0 μg/mL of OKT3diluted in phosphate-buffered saline. After overnight incubation, thesupernatants are harvested and IFN-7 in the supernatant is measured byELISA (Pierce/Endogen, Woburn, Mass.). For the coculture assay, 1×10⁵TIL cells are placed into a 96-well plate with autologous tumor cells.(1:1 ratio). After a 24-hour incubation, supernatants are harvested andIFN-7 release can be quantified, for example by ELISA.

In some embodiments, TILs that exhibit greater than 3000 pg/10⁶ TILs to300000 pg/10⁶ TILs or more Granzyme B secretion are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILS that exhibit greater than 3000 pg/10⁶ TILs greater than 5000 pg/10⁶TILs, greater than 7000 pg/10⁶ TILs, greater than 9000 pg/10⁶ TILs,greater than 11000 pg/10⁶ TILs, greater than 13000 pg/10⁶ TILs, greaterthan 15000 pg/10⁶ TILs, greater than 17000 pg/10⁶ TILs, greater than19000 pg/10⁶ TILs, greater than 20000 pg/10⁶ TILs, greater than 40000pg/10⁶ TILs, greater than 60000 pg/10⁶ TILs, greater than 80000 pg/10⁶TILs, greater than 100000 pg/10⁶ TILs, greater than 120000 pg/10⁶ TILs,greater than 140000 pg/10⁶ TILs, greater than 160000 pg/10⁶ TILs,greater than 180000 pg/10⁶ TILs, greater than 200000 pg/10⁶ TILs,greater than 220000 pg/10⁶ TILs, greater than 240000 pg/10⁶ TILs,greater than 260000 pg/10⁶ TILs, greater than 280000 pg/10⁶ TILs,greater than 300000 pg/10⁶ TILs or more Granzyme B secretion are TILsproduced by the expansion methods of the present invention, includingfor example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . Insome embodiments, TILs that exhibit greater than 3000 pg/10⁶ TILsGranzyme B secretion are TILs produced by the expansion methods of thepresent invention, including for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibit greaterthan 5000 pg/10⁶ TILs Granzyme B secretion are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 7000 pg/10⁶ TILs Granzyme B secretion areTILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 9000 pg/10⁶ TILsGranzyme B secretion are TILs produced by the expansion methods of thepresent invention, including for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibit greaterthan 11000 pg/10⁶ TILs Granzyme B secretion are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 13000 pg/10⁶ TILs Granzyme B secretionare TILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 15000 pg/10⁶TILs Granzyme B secretion are TILs produced by the expansion methods ofthe present invention, including for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibitgreater than 17000 pg/10⁶ TILs Granzyme B secretion are TILs produced bythe expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILs that exhibit greater than 19000 pg/10⁶ TILs Granzyme Bsecretion are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than20000 pg/10⁶ TILs Granzyme B secretion are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 40000 pg/10⁶ TILs Granzyme B secretionare TILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 60000 pg/10⁶TILs Granzyme B secretion are TILs produced by the expansion methods ofthe present invention, including for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibitgreater than 80000 pg/10⁶ TILs Granzyme B secretion are TILs produced bythe expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILs that exhibit greater than 100000 pg/10⁶ TILs GranzymeB secretion are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than120000 pg/10⁶ TILs Granzyme B secretion are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 140000 pg/10⁶ TILs Granzyme B secretionare TILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 160000 pg/10⁶TILs Granzyme B secretion are TILs produced by the expansion methods ofthe present invention, including for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibitgreater than 180000 pg/10⁶ TILs Granzyme B secretion are TILs producedby the expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILs that exhibit greater than 200000 pg/10⁶ TILs GranzymeB secretion are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than220000 pg/10⁶ TILs Granzyme B secretion are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 240000 pg/10⁶ TILs Granzyme B secretionare TILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 260000 pg/10⁶TILs Granzyme B secretion are TILs produced by the expansion methods ofthe present invention, including for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibitgreater than 280000 pg/10⁶ TILs Granzyme B secretion are TILs producedby the expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILs that exhibit greater than 300000 pg/10⁶ TILs GranzymeB secretion are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than 3000pg/10⁶ TILs to 300000 pg/10⁶ TILs or more Granzyme B secretion are TILsproduced by the expansion methods of the present invention, includingfor example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . Insome embodiments, TILS that exhibit greater than 3000 pg/10⁶ TILsgreater than 5000 pg/10⁶ TILs, greater than 7000 pg/10⁶ TILs, greaterthan 9000 pg/10⁶ TILs, greater than 11000 pg/10⁶ TILs, greater than13000 pg/10⁶ TILs, greater than 15000 pg/10⁶ TILs, greater than 17000pg/10⁶ TILs, greater than 19000 pg/10⁶ TILs, greater than 20000 pg/10⁶TILs, greater than 40000 pg/10⁶ TILs, greater than 60000 pg/10⁶ TILs,greater than 80000 pg/10⁶ TILs, greater than 100000 pg/10⁶ TILs, greaterthan 120000 pg/10⁶ TILs, greater than 140000 pg/10⁶ TILs, greater than160000 pg/10⁶ TILs, greater than 180000 pg/10⁶ TILs, greater than 200000pg/10⁶ TILs, greater than 220000 pg/10⁶ TILs, greater than 240000 pg/10⁶TILs, greater than 260000 pg/10⁶ TILs, greater than 280000 pg/10⁶ TILs,greater than 300000 pg/10⁶ TILs or more Granzyme B secretion are TILsproduced by the expansion methods of the present invention, includingfor example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . Insome embodiments, TILs that exhibit greater than 3000 pg/10⁶ TILsGranzyme B secretion are TILs produced by the expansion methods of thepresent invention, including for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibit greaterthan 5000 pg/10⁶ TILs Granzyme B secretion are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 7000 pg/10⁶ TILs Granzyme B secretion areTILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 9000 pg/10⁶ TILsGranzyme B secretion are TILs produced by the expansion methods of thepresent invention, including for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibit greaterthan 11000 pg/10⁶ TILs Granzyme B secretion are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 13000 pg/10⁶ TILs Granzyme B secretionare TILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 15000 pg/10⁶TILs Granzyme B secretion are TILs produced by the expansion methods ofthe present invention, including for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibitgreater than 17000 pg/10⁶ TILs Granzyme B secretion are TILs produced bythe expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILs that exhibit greater than 19000 pg/10⁶ TILs Granzyme Bsecretion are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than20000 pg/10⁶ TILs Granzyme B secretion are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 40000 pg/10⁶ TILs Granzyme B secretionare TILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 60000 pg/10⁶TILs Granzyme B secretion are TILs produced by the expansion methods ofthe present invention, including for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibitgreater than 80000 pg/10⁶ TILs Granzyme B secretion are TILs produced bythe expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILs that exhibit greater than 100000 pg/10⁶ TILs GranzymeB secretion are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than120000 pg/10⁶ TILs Granzyme B secretion are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 140000 pg/10⁶ TILs Granzyme B secretionare TILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 160000 pg/10⁶TILs Granzyme B secretion are TILs produced by the expansion methods ofthe present invention, including for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibitgreater than 180000 pg/10⁶ TILs Granzyme B secretion are TILs producedby the expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILs that exhibit greater than 200000 pg/10⁶ TILs GranzymeB secretion are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than220000 pg/10⁶ TILs Granzyme B secretion are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 240000 pg/10⁶ TILs Granzyme B secretionare TILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 260000 pg/10⁶TILs Granzyme B secretion are TILs produced by the expansion methods ofthe present invention, including for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibitgreater than 280000 pg/10⁶ TILs Granzyme B secretion are TILs producedby the expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILs that exhibit greater than 300000 pg/10⁶ TILs GranzymeB secretion are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 .

In some embodiments, TILs that exhibit greater than 1000 pg/ml to 300000pg/ml or more Granzyme B secretion are TILs produced by the expansionmethods of the present invention, including for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs thatexhibit greater than 1000 pg/ml, greater than 2000 pg/ml, greater than3000 pg/ml, greater than 4000 pg/ml, greater than 5000 pg/ml, greaterthan 6000 pg/ml, greater than 7000 pg/ml, greater than 8000 pg/ml,greater than 9000 pg/ml, greater than 10000 pg/ml, greater than 20000pg/ml, greater than 30000 pg/ml, greater than 40000 pg/ml, greater than50000 pg/ml, greater than 60000 pg/ml, greater than 70000 pg/ml, greaterthan 80000 pg/ml, greater than 90000 pg/ml, greater than 100000 pg/ml ormore Granzyme B secretion are TILs produced by the expansion methods ofthe present invention, including for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 . In some embodiments, TILS that exhibitgreater than 1000 pg/ml Granzyme B are TILs produced by the expansionmethods of the present invention, including for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs thatexhibit greater than 2000 pg/ml Granzyme B are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 3000 pg/ml Granzyme B are TILs producedby the expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILs that exhibit greater than 4000 pg/ml Granzyme B areTILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 5000 pg/mlGranzyme B are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than 6000pg/ml Granzyme B are TILs produced by the expansion methods of thepresent invention, including for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibit greaterthan 7000 pg/ml Granzyme B are TILs produced by the expansion methods ofthe present invention, including for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibitgreater than 8000 pg/ml Granzyme B are TILs produced by the expansionmethods of the present invention, including for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs thatexhibit greater than 9000 pg/ml Granzyme B are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 10000 pg/ml Granzyme B are TILs producedby the expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILS that exhibit greater than 20000 pg/ml Granzyme B areTILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 30000 pg/mlGranzyme B are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than40000 pg/ml Granzyme B are TILs produced by the expansion methods of thepresent invention, including for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibit greaterthan 50000 pg/ml Granzyme B are TILs produced by the expansion methodsof the present invention, including for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs that exhibitgreater than 60000 pg/ml Granzyme B are TILs produced by the expansionmethods of the present invention, including for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs thatexhibit greater than 70000 pg/ml Granzyme B are TILs produced by theexpansion methods of the present invention, including for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments,TILs that exhibit greater than 80000 pg/ml Granzyme B are TILs producedby the expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILs that exhibit greater than 90000 pg/ml Granzyme B areTILs produced by the expansion methods of the present invention,including for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 . In some embodiments, TILs that exhibit greater than 100000 pg/mlGranzyme B are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than120000 pg/ml Granzyme B secretion are TILs produced by the expansionmethods of the present invention, including for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs thatexhibit greater than 140000 pg/ml Granzyme B are TILs Granzyme Bsecretion are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than160000 pg/ml Granzyme B secretion are TILs produced by the expansionmethods of the present invention, including for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs thatexhibit greater than 180000 pg/ml Granzyme B secretion are TILs producedby the expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILs that exhibit greater than 200000 pg/ml Granzyme Bsecretion are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than220000 pg/ml Granzyme B secretion are TILs produced by the expansionmethods of the present invention, including for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs thatexhibit greater than 240000 pg/ml Granzyme B secretion are TILs producedby the expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 . In someembodiments, TILs that exhibit greater than 260000 pg/ml Granzyme Bsecretion are TILs produced by the expansion methods of the presentinvention, including for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 . In some embodiments, TILs that exhibit greater than280000 pg/ml Granzyme B secretion are TILs produced by the expansionmethods of the present invention, including for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 . In some embodiments, TILs thatexhibit greater than 300000 pg/ml Granzyme B secretion are TILs producedby the expansion methods of the present invention, including for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 .

In some embodiments, the expansion methods of the present inventionproduce an expanded population of TILs that exhibits increased GranzymeB secretion in vitro including for example TILs as provided in FIG. 2Aand/or FIG. 2B and/or FIG. 2C and/or FIG. 9 , as compared tonon-expanded population of TILs. In some embodiments, Granzyme Bsecretion of the expanded population of TILs of the present invention isincreased by at least one-fold to fifty-fold or more as compared tonon-expanded population of TILs. In some embodiments, IFN-γ secretion isincreased by at least one-fold, at least two-fold, at least three-fold,at least four-fold, at least five-fold, at least six-fold, at leastseven-fold, at least eight-fold, at least nine-fold, at least ten-fold,at least twenty-fold, at least thirty-fold, at least forty-fold, atleast fifty-fold or more as compared to non-expanded population of TILs.In some embodiments, Granzyme B secretion of the expanded population ofTILs of the present invention is increased by at least one-fold ascompared to non-expanded population of TILs. In some embodiments,Granzyme B secretion of the expanded population of TILs of the presentinvention is increased by at least two-fold as compared to non-expandedpopulation of TILs. In some embodiments, Granzyme B secretion of theexpanded population of TILs of the present invention is increased by atleast three-fold as compared to non-expanded population of TILs. In someembodiments, Granzyme B secretion of the expanded population of TILs ofthe present invention is increased by at least four-fold as compared tonon-expanded population of TILs. In some embodiments, Granzyme Bsecretion of the expanded population of TILs of the present invention isincreased by at least five-fold as compared to non-expanded populationof TILs. In some embodiments, Granzyme B secretion of the expandedpopulation of TILs of the present invention is increased by at leastsix-fold as compared to non-expanded population of TILs. In someembodiments, Granzyme B secretion of the expanded population of TILs ofthe present invention is increased by at least seven-fold as compared tonon-expanded population of TILs. In some embodiments, Granzyme Bsecretion of the expanded population of TILs of the present invention isincreased by at least eight-fold as compared to non-expanded populationof TILs. In some embodiments, Granzyme B secretion of the expandedpopulation of TILs of the present invention is increased by at leastnine-fold as compared to non-expanded population of TILs. In someembodiments, Granzyme B secretion of the expanded population of TILs ofthe present invention is increased by at least ten-fold as compared tonon-expanded population of TILs. In some embodiments, Granzyme Bsecretion of the expanded population of TILs of the present invention isincreased by at least twenty-fold as compared to non-expanded populationof TILs. In some embodiments, Granzyme B secretion of the expandedpopulation of TILs of the present invention is increased by at leastthirty-fold as compared to non-expanded population of TILs. In someembodiments, Granzyme B secretion of the expanded population of TILs ofthe present invention is increased by at least forty-fold as compared tonon-expanded population of TILs. In some embodiments, Granzyme Bsecretion of the expanded population of TILs of the present invention isincreased by at least fifty-fold as compared to non-expanded populationof TILs.

In some embodiments, the phenotypic characterization is examined aftercryopreservation.

J. Metabolic Health of Expanded TILs

The restimulated TILs are characterized by significant enhancement ofbasal glycolysis as compared to either freshly harvested TILs and/orpost-thawed TILs. In an embodiment, no selection of the first populationof TILs, second population of TILs, third population of TILs, harvestedTIL population, and/or the therapeutic TIL population based on CD8expression is performed during any of steps, including those discussedabove or as provided for example in FIG. 9 . In some embodiments, noselection of the first population of TILs based on CD8 expression isperformed. In some embodiments, no selection of the second population ofTILs based on CD8 expression is performed. In some embodiments, noselection of the third population of TILs based on CD8 expression isperformed. In some embodiments, no selection of the harvested populationof TILs based on CD8 expression is performed. In some embodiments, noselection of the therapeutic population of TILs based on CD8 expressionis performed.

In an embodiment, no selection of the first population of TILs, secondpopulation of TILs, third population of TILs, or harvested TILpopulation based on CD8 expression is performed during any of steps (a)to (f) of the method for expanding tumor infiltrating lymphocytes (TILs)into a therapeutic population of TILs comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T-cells and/or central memory T-cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system.

In an embodiment, no selection of the first population of TILs, secondpopulation of TILs, third population of TILs, or harvested TILpopulation based on CD8 expression is performed during any of steps (a)to (h) of the method for treating a subject with cancer, the methodcomprising administering expanded tumor infiltrating lymphocytes (TILs)comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a subject by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T-cells and/or central memory T-cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system;    -   (g) optionally cryopreserving the infusion bag comprising the        harvested TIL population from step (f) using a cryopreservation        process; and    -   (h) administering a therapeutically effective dosage of the        third population of TILs from the infusion bag in step (g) to        the patient.

The TILs prepared by the methods described herein are characterized bysignificant enhancement of basal glycolysis as compared to, for example,freshly harvested TILs and/or TILs prepared using other methods thanthose provide herein including for example, methods other than thoseembodied in FIG. 9 . In an embodiment, no selection of the firstpopulation of TILs, second population of TILs, third population of TILs,harvested TIL population, and/or the therapeutic TIL population based onCD8 expression is performed during any of steps, including thosediscussed above or as provided for example in FIG. 9 . In someembodiments, no selection of the first population of TILs based on CD8expression is performed. In some embodiments, no selection of the secondpopulation of TILs based on CD8 expression is performed. In someembodiments, no selection of the third population of TILs based on CD8expression is performed. In some embodiments, no selection of theharvested population of TILs based on CD8 expression is performed. Insome embodiments, no selection of the therapeutic population of TILsbased on CD8 expression is performed. In an embodiment, no selection ofthe first population of TILs, second population of TILs, thirdpopulation of TILs, or harvested TIL population based on CD8 expressionis performed during any of steps (a) to (h).

Spare respiratory capacity (SRC) and glycolytic reserve can be evaluatedfor TILs expanded with different methods of the present disclosure. TheSeahorse XF Cell Mito Stress Test measures mitochondrial function bydirectly measuring the oxygen consumption rate (OCR) of cells, usingmodulators of respiration that target components of the electrontransport chain in the mitochondria. The test compounds (oligomycin,FCCP, and a mix of rotenone and antimycin A, described below) areserially injected to measure ATP production, maximal respiration, andnon-mitochondrial respiration, respectively. Proton leak and sparerespiratory capacity are then calculated using these parameters andbasal respiration. Each modulator targets a specific component of theelectron transport chain. Oligomycin inhibits ATP synthase (complex V)and the decrease in OCR following injection of oligomycin correlates tothe mitochondrial respiration associated with cellular ATP production.Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) is anuncoupling agent that collapses the proton gradient and disrupts themitochondrial membrane potential. As a result, electron flow through theelectron transport chain is uninhibited and oxygen is maximally consumedby complex IV. The FCCP-stimulated OCR can then be used to calculatespare respiratory capacity, defined as the difference between maximalrespiration and basal respiration. Spare respiratory capacity (SRC) is ameasure of the ability of the cell to respond to increased energydemand. The third injection is a mix of rotenone, a complex I inhibitor,and antimycin A, a complex III inhibitor. This combination shuts downmitochondrial respiration and enables the calculation ofnonmitochondrial respiration driven by processes outside themitochondria. In some embodiments, the comparison is to, for example,freshly harvested TILs and/or TILs prepared using other methods thanthose provide herein including for example, methods other than thoseembodied in FIG. 9 .

In some embodiments, the metabolic assay is basal respiration. Ingeneral, second expansion TILs have a basal respiration rate that is atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% of the basal respiration rateof freshly harvested TILs and/or TILs prepared using other methods thanthose provide herein including for example, methods other than thoseembodied in FIG. 9 . In some embodiments, the basal respiration rate isfrom about 50% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 9 . In some embodiments, the basal respiration rate is fromabout 60% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 9 . In some embodiments, the basal respiration rate is fromabout 70% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 9 . In some embodiments, the basal respiration rate is fromabout 80% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 9 . In some embodiments, the basal respiration rate is fromabout 90% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 9 . In some embodiments, the basal respiration rate is fromabout 95% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 9 . In some embodiments, the second expansion TILs or secondadditional expansion TILs (such as, for example, those described in StepD of FIG. 9 , including TILs referred to as reREP TILs) have a basalrespiration rate that is not statistically significantly different thanthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the comparison is to, for example, freshly harvested TILsand/or TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 9 .

In some embodiments, the metabolic assay is spare respiratory capacity.In general, the second expansion TILs or second additional expansionTILs (such as, for example, those described in Step D of FIG. 9 ,including TILs referred to as reREP TILs) have a spare respiratorycapacity that is at least is at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, at least 98%, or at least 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the spare respiratory capacity is from about 50% to about99% of the basal respiration rate of freshly harvested TILs. In someembodiments, the spare respiratory capacity is from about 50% to about99% of the basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the spare respiratory capacity is from about 60% to about99% of the basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the spare respiratory capacity is from about 70% to about99% of the basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the spare respiratory capacity is from about 80% to about99% of the basal respiration rate of freshly harvested TILs. In someembodiments, the spare respiratory capacity is from about 90% to about99% of the basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the spare respiratory capacity is from about 95% to about99% of the basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the second expansion TILs or second additional expansionTILs (such as, for example, those described in Step D of FIG. 9 ,including TILs referred to as reREP TILs) have a spare respiratorycapacity that is not statistically significantly different than thebasal respiration rate of freshly harvested TILs and/or TILs preparedusing other methods than those provide herein including for example,methods other than those embodied in FIG. 9 .

In general, second expansion TILs or second additional expansion TILs(such as, for example, those described in Step D of FIG. 9 , includingTILs referred to as reREP TILs) have a spare respiratory capacity thatis at least is at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 98%, or at least 99% of the basalrespiration rate of freshly harvested TILs and/or TILs prepared usingother methods than those provide herein including for example, methodsother than those embodied in FIG. 9 . In some embodiments, the metabolicassay measured is glycolytic reserve. In some embodiments, the metabolicassay is spare respiratory capacity. To measure cellular (respiratory)metabolism cells were treated with inhibitors of mitochondrialrespiration and glycolysis to determine a metabolic profile for the TILconsisting of the following measures: baseline oxidative phosphorylation(as measured by OCR), spare respiratory capacity, baseline glycolyticactivity (as measured by ECAR), and glycolytic reserve. Metabolicprofiles were performed using the Seahorse CombinationMitochondrial/Glycolysis Stress Test Assay (including the kitcommercially available from Agilent®), which allows for determining acells' capacity to perform glycolysis upon blockage of mitochondrial ATPproduction. In some embodiments, cells are starved of glucose, thenglucose is injected, followed by a stress agent. In some embodiments,the stress agent is selected from the group consisting of oligomycin,FCCP, rotenone, antimycin A and/or 2-deoxyglucose (2-DG), as well ascombinations thereof. In some embodiments, oligomycin is added at 10 mM.In some embodiments, FCCP is added at 10 mM. In some embodiments,rotenone is added at 2.5 mM. In some embodiments, antimycin A is addedat 2.5 mM. In some embodiments, 2-deoxyglucose (2-DG) is added at 500mM. In some embodiments, glycolytic capacity, glycolytic reserve, and/ornon-glycolytic acidification are measured. In general, TILs have aglycolytic reserve that is at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, at least 98%, or at least 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the glycolytic reserve is from about 50% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the glycolytic reserve is from about 60% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the glycolytic reserve is from about 70% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the glycolytic reserve is from about 80% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the glycolytic reserve is from about 90% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the glycolytic reserve is from about 95% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 .

In some embodiments, the metabolic assay is basal glycolysis. In someembodiments, second expansion TILs or second additional expansion TILs(such as, for example, those described in Step D of FIG. 9 , includingTILs referred to as reREP TILs) have an increase in basal glycolysis ofat least two-fold, at least three-fold, at least four-fold, at leastfive-fold, at least six-fold, at least 7-fold, at least eight-fold, atleast nine-fold, or at least ten-fold as compared to freshly harvestedTILs and/or TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 9 . Insome embodiments, the second expansion TILs or second additionalexpansion TILs (such as, for example, those described in Step D of FIG.9 , including TILs referred to as reREP TILs) have an increase in basalglycolysis of about two-fold to about ten-fold as compared to freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 9 . In some embodiments, the second expansion TILs or secondadditional expansion TILs (such as, for example, those described in StepD of FIG. 9 , including TILs referred to as reREP TILs) have an increasein basal glycolysis of about two-fold to about eight-fold as compared tofreshly harvested TILs and/or TILs prepared using other methods thanthose provide herein including for example, methods other than thoseembodied in FIG. 9 . In some embodiments, second expansion TILs orsecond additional expansion TILs (such as, for example, those describedin Step D of FIG. 9 , including TILs referred to as reREP TILs) have anincrease in basal glycolysis of about three-fold to about seven-fold ascompared to freshly harvested TILs and/or TILs prepared using othermethods than those provide herein including for example, methods otherthan those embodied in FIG. 9 . In some embodiments, the secondexpansion TILs or second additional expansion TILs (such as, forexample, those described in Step D of FIG. 9 , including TILs referredto as reREP TILs) have an increase in basal glycolysis of about two-foldto about four-fold as compared to freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the second expansion TILs or second additional expansionTILs (such as, for example, those described in Step D of FIG. 9 ,including TILs referred to as reREP TILs) have an increase in basalglycolysis of about two-fold to about three-fold as compared to freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 9 .

In general, the second expansion TILs or second additional expansionTILs (such as, for example, those described in Step D of FIG. 9 ,including TILs referred to as reREP TILs) have a glycolytic reserve thatis at least 50%, at least 55%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% of the basal respiration rateof freshly harvested TILs and/or TILs prepared using other methods thanthose provide herein including for example, methods other than thoseembodied in FIG. 9 . In some embodiments, the glycolytic reserve is fromabout 50% to about 99% of the basal respiration rate of freshlyharvested TILs. In some embodiments, the glycolytic reserve is fromabout 60% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 9 . In some embodiments, the glycolytic reserve is from about70% to about 99% of the basal respiration rate of freshly harvested TILsand/or TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 9 . Insome embodiments, the glycolytic reserve is from about 80% to about 99%of the basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the glycolytic reserve is from about 90% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . In someembodiments, the glycolytic reserve is from about 95% to about 99% ofthe basal respiration rate of freshly harvested TILs.

Granzyme B Production: Granzyme B is another measure of the ability ofTIL to kill target cells. Media supernatants restimulated as describedabove using antibodies to CD3, CD28, and CD137/4-1BB were also evaluatedfor their levels of Granzyme B using the Human Granzyme B DuoSet ELISAKit (R & D Systems, Minneapolis, Minn.) according to the manufacturer'sinstructions. In some embodiments, the second expansion TILs or secondadditional expansion TILs (such as, for example, those described in StepD of FIG. 9 , including TILs referred to as reREP TILs) have increasedGranzyme B production. In some embodiments, the second expansion TILs orsecond additional expansion TILs (such as, for example, those describedin Step D of FIG. 9 , including TILs referred to as reREP TILs) haveincreased cytotoxic activity.

In some embodiments, telomere length can be used as a measure of cellviability and/or cellular function. In some embodiments, the telomeresare surprisingly the same length in the TILs produced by the presentinvention as compared to TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 9 . Telomere length measurement: Diverse methods have been usedto measure the length of telomeres in genomic DNA and cytologicalpreparations. The telomere restriction fragment (TRF) analysis is thegold standard to measure telomere length (de Lange et al., 1990).However, the major limitation of TRF is the requirement of a largeamount of DNA (1.5{circumflex over ( )}g). Two widely used techniquesfor the measurement of telomere lengths namely, fluorescence in situhybridization (FISH; Agilent Technologies, Santa Clara, Calif.) andquantitative PCR can be employed with the present invention. In someembodiments, there is no change in telomere length between the initiallyharvest TILs in Step A and the expanded TILs from for example Step D asprovided in FIG. 9 .

In some embodiments, the TILs express one more markers selected from thegroup consisting of granzyme B, perforin, and granulysin. In someembodiments, the TILs express granzyme B. In some embodiments, the TILsexpress perforin. In some embodiments, the TILs express granulysin.

In an embodiment, restimulated TILs can also be evaluated for cytokinerelease, using cytokine release assays. In some embodiments, TILs can beevaluated for interferon-γ (IFN-γ) secretion. In some embodiments, theIFN-γ secretion is measured by an ELISA assay. In some embodiments, theIFN-γ secretion is measured by an ELISA assay after the rapid secondexpansion step, after Step D as provided in for example, FIG. 2 (inparticular, e.g., FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 ).In some embodiments, TIL health is measured by IFN-gamma (IFN-γ)secretion. In some embodiments, IFN-γ secretion is indicative of activeTILs. In some embodiments, a potency assay for IFN-γ production isemployed. IFN-γ production is another measure of cytotoxic potential.IFN-γ production can be measured by determining the levels of thecytokine IFN-γ in the media of TIL stimulated with antibodies to CD3,CD28, and CD137/4-1BB. IFN-γ levels in media from these stimulated TILcan be determined using by measuring IFN-γ release. In some embodiments,an increase in IFN-γ production in for example Step D in the Gen 3process as provided in FIG. 2 (in particular, e.g., FIG. 2A and/or FIG.2B and/or FIG. 2C and/or FIG. 9 ) TILs as compared to for example Step Din the 2A process as provided in FIG. 2 (in particular, e.g., FIG. 2A)is indicative of an increase in cytotoxic potential of the Step D TILs.In some embodiments, IFN-γ secretion is increased one-fold, two-fold,three-fold, four-fold, or five-fold or more. In some embodiments, IFN-γsecretion is increased one-fold. In some embodiments, IFN-γ secretion isincreased two-fold. In some embodiments, IFN-γ secretion is increasedthree-fold. In some embodiments, IFN-γ secretion is increased four-fold.In some embodiments, IFN-γ secretion is increased five-fold. In someembodiments, IFN-γ is measured using a Quantikine ELISA kit. In someembodiments, IFN-γ is measured in TILs ex vivo. In some embodiments,IFN-γ is measured in TILs ex vivo, including TILs produced by themethods of the present invention, including, for example FIG. 2Bmethods.

In some embodiments, TILs capable of at least one-fold, two-fold,three-fold, four-fold, or five-fold or more IFN-γ secretion are TILsproduced by the expansion methods of the present invention, including,for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.In some embodiments, TILs capable of at least one-fold more IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at leasttwo-fold more IFN-γ secretion are TILs produced by the expansion methodsof the present invention, including, for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILs capableof at least three-fold more IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least four-fold more IFN-γ secretion areTILs produced by the expansion methods of the present invention,including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 methods. In some embodiments, TILs capable of at least five-fold moreIFN-γ secretion are TILs produced by the expansion methods of thepresent invention, including, for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 methods.

In some embodiments, TILs capable of at least 100 pg/ml to about 1000pg/mL or more IFN-γ secretion are TILs produced by the expansion methodsof the present invention, including, for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILs capableof at least 200 pg/ml, at least 250 pg/ml, at least 300 pg/ml, at least350 pg/ml, at least 400 pg/ml, at least 450 pg/ml, at least 500 pg/ml,at least 550 pg/ml, at least 600 pg/ml, at least 650 pg/ml, at least 700pg/ml, at least 750 pg/ml, at least 800 pg/ml, at least 850 pg/ml, atleast 900 pg/ml, at least 950 pg/ml, or at least 1000 pg/mL or moreIFN-γ secretion are TILs produced by the expansion methods of thepresent invention, including, for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 methods. In some embodiments, TILs capable of atleast 200 pg/ml IFN-γ secretion are TILs produced by the expansionmethods of the present invention, including, for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILscapable of at least 200 pg/ml IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 300 pg/ml IFN-γ secretion are TILsproduced by the expansion methods of the present invention, including,for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.In some embodiments, TILs capable of at least 400 pg/ml IFN-γ secretionare TILs produced by the expansion methods of the present invention,including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 methods. In some embodiments, TILs capable of at least 500 pg/ml IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at least 600pg/ml IFN-γ secretion are TILs produced by the expansion methods of thepresent invention, including, for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 methods. In some embodiments, TILs capable of atleast 700 pg/ml IFN-γ secretion are TILs produced by the expansionmethods of the present invention, including, for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILscapable of at least 800 pg/ml IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 900 pg/ml IFN-γ secretion are TILsproduced by the expansion methods of the present invention, including,for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.In some embodiments, TILs capable of at least 1000 pg/ml IFN-γ secretionare TILs produced by the expansion methods of the present invention,including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 methods. In some embodiments, TILs capable of at least 2000 pg/mlIFN-γ secretion are TILs produced by the expansion methods of thepresent invention, including, for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 methods. In some embodiments, TILs capable of atleast 3000 pg/ml IFN-γ secretion are TILs produced by the expansionmethods of the present invention, including, for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILscapable of at least 4000 pg/ml IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 5000 pg/ml IFN-γ secretion areTILs produced by the expansion methods of the present invention,including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 methods. In some embodiments, TILs capable of at least 6000 pg/mlIFN-γ secretion are TILs produced by the expansion methods of thepresent invention, including, for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 methods. In some embodiments, TILs capable of atleast 7000 pg/ml IFN-γ secretion are TILs produced by the expansionmethods of the present invention, including, for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILscapable of at least 8000 pg/ml IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 9000 pg/ml IFN-γ secretion areTILs produced by the expansion methods of the present invention,including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 methods. In some embodiments, TILs capable of at least 10,000 pg/mlIFN-γ secretion are TILs produced by the expansion methods of thepresent invention, including, for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 methods. In some embodiments, TILs capable of atleast 15,000 pg/ml IFN-γ secretion are TILs produced by the expansionmethods of the present invention, including, for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILscapable of at least 20,000 pg/ml IFN-γ secretion are TILs produced bythe expansion methods of the present invention, including, for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 25,000 pg/ml IFN-γ secretion areTILs produced by the expansion methods of the present invention,including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 methods. In some embodiments, TILs capable of at least 30,000 pg/mlIFN-γ secretion are TILs produced by the expansion methods of thepresent invention, including, for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 methods. In some embodiments, TILs capable of atleast 35,000 pg/ml IFN-γ secretion are TILs produced by the expansionmethods of the present invention, including, for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILscapable of at least 40,000 pg/ml IFN-γ secretion are TILs produced bythe expansion methods of the present invention, including, for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 45,000 pg/ml IFN-γ secretion areTILs produced by the expansion methods of the present invention,including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 methods. In some embodiments, TILs capable of at least 50,000 pg/mlIFN-γ secretion are TILs produced by the expansion methods of thepresent invention, including, for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 methods.

In some embodiments, TILs capable of at least 100 pg/ml/5e5 cells toabout 1000 pg/ml/5e5 cells or more IFN-γ secretion are TILs produced bythe expansion methods of the present invention, including, for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 200 pg/ml/5e5 cells, at least 250pg/ml/5e5 cells, at least 300 pg/ml/5e5 cells, at least 350 pg/ml/5e5cells, at least 400 pg/ml/5e5 cells, at least 450 pg/ml/5e5 cells, atleast 500 pg/ml/5e5 cells, at least 550 pg/ml/5e5 cells, at least 600pg/ml/5e5 cells, at least 650 pg/ml/5e5 cells, at least 700 pg/ml/5e5cells, at least 750 pg/ml/5e5 cells, at least 800 pg/ml/5e5 cells, atleast 850 pg/ml/5e5 cells, at least 900 pg/ml/5e5 cells, at least 950pg/ml/5e5 cells, or at least 1000 pg/ml/5e5 cells or more IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at least 200pg/ml/5e5 cells IFN-γ secretion are TILs produced by the expansionmethods of the present invention, including, for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILscapable of at least 200 pg/ml/5e5 cells IFN-γ secretion are TILsproduced by the expansion methods of the present invention, including,for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.In some embodiments, TILs capable of at least 300 pg/ml/5e5 cells IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at least 400pg/ml/5e5 cells IFN-γ secretion are TILs produced by the expansionmethods of the present invention, including, for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILscapable of at least 500 pg/ml/5e5 cells IFN-γ secretion are TILsproduced by the expansion methods of the present invention, including,for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.In some embodiments, TILs capable of at least 600 pg/ml/5e5 cells IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at least 700pg/ml/5e5 cells IFN-γ secretion are TILs produced by the expansionmethods of the present invention, including, for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILscapable of at least 800 pg/ml/5e5 cells IFN-γ secretion are TILsproduced by the expansion methods of the present invention, including,for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.In some embodiments, TILs capable of at least 900 pg/ml/5e5 cells IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at least1000 pg/ml/5e5 cells IFN-γ secretion are TILs produced by the expansionmethods of the present invention, including, for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILscapable of at least 2000 pg/ml/5e5 cells IFN-γ secretion are TILsproduced by the expansion methods of the present invention, including,for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.In some embodiments, TILs capable of at least 3000 pg/ml/5e5 cells IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at least4000 pg/ml/5e5 cells IFN-γ secretion are TILs produced by the expansionmethods of the present invention, including, for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILscapable of at least 5000 pg/ml/5e5 cells IFN-γ secretion are TILsproduced by the expansion methods of the present invention, including,for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.In some embodiments, TILs capable of at least 6000 pg/ml/5e5 cells IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at least7000 pg/ml/5e5 cells IFN-γ secretion are TILs produced by the expansionmethods of the present invention, including, for example FIG. 2A and/orFIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In some embodiments, TILscapable of at least 8000 pg/ml/5e5 cells IFN-γ secretion are TILsproduced by the expansion methods of the present invention, including,for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.In some embodiments, TILs capable of at least 9000 pg/ml/5e5 cells IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at least10,000 pg/ml/5e5 cells IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 15,000 pg/ml/5e5 cells IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at least20,000 pg/ml/5e5 cells IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 25,000 pg/ml/5e5 cells IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at least30,000 pg/ml/5e5 cells IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 35,000 pg/ml/5e5 cells IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at least40,000 pg/ml/5e5 cells IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 45,000 pg/ml/5e5 cells IFN-γsecretion are TILs produced by the expansion methods of the presentinvention, including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2Cand/or FIG. 9 methods. In some embodiments, TILs capable of at least50,000 pg/ml/5e5 cells IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.

In some embodiments, TILs capable of at least one-fold, two-fold,three-fold, four-fold, or five-fold or more lower levels of TNF-α (i.e.,TNF-alpha) secretion as compared to IFN-γ secretion are TILs produced bythe expansion methods of the present invention, including, for exampleFIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least one-fold lower levels of TNF-αsecretion as compared to IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least two-fold lower levels of TNF-αsecretion as compared to IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least three-fold lower levels of TNF-αsecretion as compared to IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least four-fold lower levels of TNF-αsecretion as compared to IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least five-fold lower levels of TNF-αsecretion as compared to IFN-γ secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.

In some embodiments, TILs capable of at least 200 pg/ml/5e5 cells toabout 10,000 pg/ml/5e5 cells or more TNF-α (i.e., TNF-alpha) secretionare TILs produced by the expansion methods of the present invention,including, for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG.9 methods. In some embodiments, TILs capable of at least 500 pg/ml/5e5cells to about 10,000 pg/ml/5e5 cells or more TNF-α secretion are TILsproduced by the expansion methods of the present invention, including,for example FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.In some embodiments, TILs capable of at least 1000 pg/ml/5e5 cells toabout 10,000 pg/ml/5e5 cells or more TNF-α secretion are TILs producedby the expansion methods of the present invention, including, forexample FIG. 2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. Insome embodiments, TILs capable of at least 2000 pg/ml/5e5 cells to about10,000 pg/ml/5e5 cells or more TNF-α secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 3000 pg/ml/5e5 cells to about10,000 pg/ml/5e5 cells or more TNF-α secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 4000 pg/ml/5e5 cells to about10,000 pg/ml/5e5 cells or more TNF-α secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 5000 pg/ml/5e5 cells to about10,000 pg/ml/5e5 cells or more TNF-α secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 6000 pg/ml/5e5 cells to about10,000 pg/ml/5e5 cells or more TNF-α secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 7000 pg/ml/5e5 cells to about10,000 pg/ml/5e5 cells or more TNF-α secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 8000 pg/ml/5e5 cells to about10,000 pg/ml/5e5 cells or more TNF-α secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, TILs capable of at least 9000 pg/ml/5e5 cells to about10,000 pg/ml/5e5 cells or more TNF-α secretion are TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods.

In some embodiments, IFN-γ and granzyme B levels are measured todetermine the phenotypic characteristics of the TILs produced by theexpansion methods of the present invention, including, for example FIG.2A and/or FIG. 2B and/or FIG. 2C and/or FIG. 9 methods. In someembodiments, IFN-γ and TNF-α levels are measured to determine thephenotypic characteristics of the TILs produced by the expansion methodsof the present invention, including, for example FIG. 2A and/or FIG. 2Band/or FIG. 2C and/or FIG. 9 methods. In some embodiments, granzyme Band TNF-α levels are measured to determine the phenotypiccharacteristics of the TILs produced by the expansion methods of thepresent invention, including, for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 methods. In some embodiments, IFN-γ, granzyme Band TNF-α levels are measured to determine the phenotypiccharacteristics of the TILs produced by the expansion methods of thepresent invention, including, for example FIG. 2A and/or FIG. 2B and/orFIG. 2C and/or FIG. 9 methods.

In some embodiments, the cytotoxic potential of TIL to lyse target cellswas assessed using a co-culture assay of TIL with the bioluminescentcell line, P815 (Clone G6), according to a bioluminescent redirectedlysis assay (potency assay) for TIL assay which measures TILcytotoxicity in a highly sensitive dose dependent manner.

In some embodiments, the present methods provide an assay for assessingTIL viability, using the methods as described above. In someembodiments, the TILs are expanded as discussed above, including forexample as provided in FIG. 9 . In some embodiments, the TILs arecryopreserved prior to being assessed for viability. In someembodiments, the viability assessment includes thawing the TILs prior toperforming a first expansion, a second expansion, and an additionalsecond expansion. In some embodiments, the present methods provide anassay for assessing cell proliferation, cell toxicity, cell death,and/or other terms related to viability of the TIL population. Viabilitycan be measured by any of the TIL metabolic assays described above aswell as any methods know for assessing cell viability that are known inthe art. In some embodiments, the present methods provide as assay forassessment of cell proliferation, cell toxicity, cell death, and/orother terms related to viability of the TILs expanded using the methodsdescribed herein, including those exemplified in FIG. 9 .

The present invention also provides assay methods for determining TILviability. In some embodiments, the TILs have equal viability ascompared to freshly harvested TILs and/or TILs prepared using othermethods than those provide herein including for example, methods otherthan those embodied in FIG. 9 . In some embodiments, the TILs haveincreased viability as compared to freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9 . The presentdisclosure provides methods for assaying TILs for viability by expandingtumor infiltrating lymphocytes (TILs) into a larger population of TILscomprising:

(i) obtaining a first population of TILs which has been previouslyexpanded;

(ii) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 to produce a secondpopulation of TILs; and

(iii) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, OKT-3, andantigen presenting cells (APCs), to produce a third population of TILs,wherein the third population of TILs is at least 100-fold greater innumber than the second population of TILs, and wherein the secondexpansion is performed for at least 14 days in order to obtain the thirdpopulation of TILs, wherein the third population of TILs comprises anincreased subpopulation of effector T-cells and/or central memoryT-cells relative to the second population of TILs, and wherein the thirdpopulation is further assayed for viability.

In some embodiments, the method further comprises:

(iv) performing an additional second expansion by supplementing the cellculture medium of the third population of TILs with additional IL-2,additional OKT-3, and additional APCs, wherein the additional secondexpansion is performed for at least 14 days to obtain a largerpopulation of TILs than obtained in step (iii), wherein the largerpopulation of TILs comprises an increased subpopulation of effectorT-cells and/or central memory T-cells relative to the third populationof TILs, and wherein the third population is further assayed forviability.

In some embodiments, prior to step (i), the cells are cryopreserved.

In some embodiments, the cells are thawed prior to performing step (i).

In some embodiments, step (iv) is repeated one to four times in order toobtain sufficient TILs for analysis.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 40 days to about 50 days.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 42 days to about 48 days.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 42 days to about 45 days.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin about 44 days.

In some embodiments, the cells from steps (iii) or (iv) express CD4,CD8, and TCR α β at levels similar to freshly harvested cells.

In some embodiments, the antigen presenting cells are peripheral bloodmononuclear cells (PBMCs).

In some embodiments, the PBMCs are added to the cell culture on any ofdays 9 through 17 in step (iii).

In some embodiments, the effector T-cells and/or central memory T-cellsin the larger population of TILs in step (iv) exhibit one or morecharacteristics selected from the group consisting of expression ofCD27, expression of CD28, longer telomeres, increased CD57 expression,and decreased CD56 expression, relative to effector T-cells, and/orcentral memory T-cells in the third population of cells.

In some embodiments, the effector T-cells and/or central memory T-cellsexhibit increased CD57 expression and decreased CD56 expression.

In some embodiments, the APCs are artificial APCs (aAPCs).

In some embodiments, the method further comprises the step oftransducing the first population of TILs with an expression vectorcomprising a nucleic acid encoding a high-affinity T-cell receptor.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the method further comprises the step oftransducing the first population of TILs with an expression vectorcomprising a nucleic acid encoding a chimeric antigen receptor (CAR)comprising a single chain variable fragment antibody fused with at leastone endodomain of a T-cell signaling molecule.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the TILs are assayed for viability.

In some embodiments, the TILs are assayed for viability aftercryopreservation.

In some embodiments, the TILs are assayed for viability aftercryopreservation and after step (iv).

The diverse antigen receptors of T and B lymphocytes are produced bysomatic recombination of a limited, but large number of gene segments.These gene segments: V (variable), D (diversity), J (joining), and C(constant), determine the binding specificity and downstreamapplications of immunoglobulins and T-cell receptors (TCRs). The presentinvention provides a method for generating TILs which exhibit andincrease the T-cell repertoire diversity (sometimes referred to aspolyclonality). In some embodiments, the increase in T-cell repertoirediversity is as compared to freshly harvested TILs and/or TILs preparedusing other methods than those provide herein including for example,methods other than those embodied in FIG. 9 . In some embodiments, theTILs obtained by the present method exhibit an increase in the T-cellrepertoire diversity. In some embodiments, the TILs obtained in thefirst expansion exhibit an increase in the T-cell repertoire diversity.In some embodiments, the increase in diversity is an increase in theimmunoglobulin diversity and/or the T-cell receptor diversity. In someembodiments, the diversity is in the immunoglobulin is in theimmunoglobulin heavy chain. In some embodiments, the diversity is in theimmunoglobulin is in the immunoglobulin light chain. In someembodiments, the diversity is in the T-cell receptor. In someembodiments, the diversity is in one of the T-cell receptors selectedfrom the group consisting of alpha, beta, gamma, and delta receptors. Insome embodiments, there is an increase in the expression of T-cellreceptor (TCR) alpha and/or beta. In some embodiments, there is anincrease in the expression of T-cell receptor (TCR) alpha. In someembodiments, there is an increase in the expression of T-cell receptor(TCR) beta. In some embodiments, there is an increase in the expressionof TCRab (i.e., TCRα/β).

According to the present disclosure, a method for assaying TILs forviability and/or further use in administration to a subject. In someembodiments, the method for assay tumor infiltrating lymphocytes (TILs)comprises:

(i) obtaining a first population of TILs;

(ii) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 to produce a secondpopulation of TILs; and

(iii) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, OKT-3, andantigen presenting cells (APCs), to produce a third population of TILs,wherein the third population of TILs is at least 50-fold greater innumber than the second population of TILs;(iv) harvesting, washing, and cryopreserving the third population ofTILs;(v) storing the cryopreserved TILs at a cryogenic temperature;(vi) thawing the third population of TILs to provide a thawed thirdpopulation of TILs; and(vii) performing an additional second expansion of a portion of thethawed third population of TILs by supplementing the cell culture mediumof the third population with IL-2, OKT-3, and APCs for an additionalexpansion period (sometimes referred to as a reREP period) of at least 3days, wherein the third expansion is performed to obtain a fourthpopulation of TILs, wherein the number of TILs in the fourth populationof TILs is compared to the number of TILs in the third population ofTILs to obtain a ratio;(viii) determining based on the ratio in step (vii) whether the thawedpopulation of TILs is suitable for administration to a patient;(ix) administering a therapeutically effective dosage of the thawedthird population of TILs to the patient when the ratio of the number ofTILs in the fourth population of TILs to the number of TILs in the thirdpopulation of TILs is determined to be greater than 5:1 in step (viii).

In some embodiments, the additional expansion period (sometimes referredto as a reREP period) is performed until the ratio of the number of TILsin the fourth population of TILs to the number of TILs in the thirdpopulation of TILs is greater than 50:1.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×10¹⁰ to about 13.7×10¹⁰.

In some embodiments, steps (i) through (vii) are performed within aperiod of about 40 days to about 50 days. In some embodiments, steps (i)through (vii) are performed within a period of about 42 days to about 48days. In some embodiments, steps (i) through (vii) are performed withina period of about 42 days to about 45 days. In some embodiments, steps(i) through (vii) are performed within about 44 days.

In some embodiments, the cells from steps (iii) or (vii) express CD4,CD8, and TCR α β at levels similar to freshly harvested cells. In someembodiments the cells are TILs.

In some embodiments, the antigen presenting cells are peripheral bloodmononuclear cells (PBMCs). In some embodiments, the PBMCs are added tothe cell culture on any of days 9 through 17 in step (iii).

In some embodiments, the effector T-cells and/or central memory T-cellsin the larger population of TILs in steps (iii) or (vii) exhibit one ormore characteristics selected from the group consisting of expression ofCD27, expression of CD28, longer telomeres, increased CD57 expression,and decreased CD56 expression, relative to effector T-cells, and/orcentral memory T-cells in the third population of cells.

In some embodiments, the effector T-cells and/or central memory T-cellsexhibit increased CD57 expression and decreased CD56 expression.

In some embodiments, the APCs are artificial APCs (aAPCs).

In some embodiments, the step of transducing the first population ofTILs with an expression vector comprising a nucleic acid encoding ahigh-affinity T-cell receptor.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the step of transducing the first population ofTILs with an expression vector comprising a nucleic acid encoding achimeric antigen receptor (CAR) comprising a single chain variablefragment antibody fused with at least one endodomain of a T-cellsignaling molecule.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the TILs are assayed for viability after step(vii).

The present disclosure also provides further methods for assaying TILs.In some embodiments, the disclosure provides a method for assaying TILscomprising:

(i) obtaining a portion of a first population of cryopreserved TILs;

(ii) thawing the portion of the first population of cryopreserved TILs;

(iii) performing a first expansion by culturing the portion of the firstpopulation of TILs in a cell culture medium comprising IL-2, OKT-3, andantigen presenting cells (APCs) for an additional expansion period(sometimes referred to as a reREP period) of at least 3 days, to producea second population of TILs, wherein the portion from the firstpopulation of TILs is compared to the second population of TILs toobtain a ratio of the number of TILs, wherein the ratio of the number ofTILs in the second population of TILs to the number of TILs in theportion of the first population of TILs is greater than 5:1;(iv) determining based on the ratio in step (iii) whether the firstpopulation of TILs is suitable for use in therapeutic administration toa patient;(v) determining the first population of TILs is suitable for use intherapeutic administration when the ratio of the number of TILs in thesecond population of TILs to the number of TILs in the first populationof TILs is determined to be greater than 5:1 in step (iv).

In some embodiments, the ratio of the number of TILs in the secondpopulation of TILs to the number of TILs in the portion of the firstpopulation of TILs is greater than 50:1.

In some embodiments, the method further comprises performing expansionof the entire first population of cryopreserved TILs from step (i)according to the methods as described in any of the embodiments providedherein.

In some embodiments, the method further comprises administering theentire first population of cryopreserved TILs from step (i) to thepatient.

K. Closed Systems for TIL Manufacturing

The present invention provides for the use of closed systems during theTIL culturing process. Such closed systems allow for preventing and/orreducing microbial contamination, allow for the use of fewer flasks, andallow for cost reductions. In some embodiments, the closed system usestwo containers.

Such closed systems are well-known in the art and can be found, forexample, at http://www.fda.gov/cber/guidelines.htm andhttps://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm.

As provided on the FDA website, closed systems with sterile methods areknown and well described. See,https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm,as referenced above and provided in pertinent part below.

Introduction

Sterile connecting devices (STCDs) produce sterile welds between twopieces of compatible tubing. This procedure permits sterile connectionof a variety of containers and tube diameters. This guidance describesrecommended practices and procedures for use of these devices. Thisguidance does not address the data or information that a manufacturer ofa sterile connecting device must submit to FDA in order to obtainapproval or clearance for marketing. It is also important to note thatthe use of an approved or cleared sterile connecting device for purposesnot authorized in the labeling may cause the device to be consideredadulterated and misbranded under the Federal Food, Drug and CosmeticAct.

1. FDA Recommendations

Manufacturers of blood products who propose to routinely use anFDA-cleared STCD should incorporate information regarding such use instandard operating procedure (SOP) manuals for each blood product. Theseentries should include record keeping, product tracking, tube weldquality control, lot numbers of software and disposables (includingsource(s) of elements to be added). Quality control procedures shouldinclude a test of the integrity of each weld.

2. Applications of the STCD

The user should be aware that use of the device may create a new productor significantly modify the configuration of a regulated product forwhich safety and efficacy have not been demonstrated. For those “newproducts” subject to licensure, applications, or application supplementsmust be submitted to FDA in addition to submission of a SOP. In general,pooling or mixing that involves cellular components represents a changein the product that requires submission and approval of a licenseapplication or application supplement. Such applications and applicationsupplements should contain data and descriptions of manufacturingprocedures that demonstrate that the “new product” is safe and effectivefor its intended use throughout the proposed dating period.

The following comments are provided as guidance on the more common usesof an FDA cleared or approved STCD:

L. Adding a new or smaller needle to a blood collection set

Using the STCD to add a needle prior to the initiation of a procedure(whole blood collection, plateletpheresis or source plasma collection)is not considered to open a functionally closed system. If a needle isadded during a procedure, only an STCD approved to weld liquid-filledtubing should be used. If the test of weld integrity is satisfactory,the use of an STCD is not considered to open a functionally closedsystem.

Platelets, Pheresis prepared in an open system should be labeled with a24 hour outdate and Platelets, Pheresis products prepared in afunctionally closed system should be labeled with a five day outdate(See Revised Guideline for Collection of Platelets, Pheresis, Oct. 7,1988).

The source and specifications of added tubing and needles should beaddressed in the blood center's SOP and records. Using the STCD to addneedles does not represent a major change in manufacturing for whichlicensed establishments need preapproval.

M. Using the STCD to Prepare Components

When the STCD is used to attach additional component preparation bags,records should be properly maintained identifying the source of thetransfer packs and the appropriate verification of blood unit number andABO/Rh. All blood and blood components must be appropriately labeled (21CFR 606.121).

Examples

-   -   Adding a fourth bag to a whole blood collection triple-pack for        the production of Cryoprecipitated AHF from Fresh Frozen Plasma.    -   Connection of an additive solution to a red blood cell unit.    -   Addition of an in-line filter that has been FDA cleared for use        in manufacturing components.    -   Addition of a third storage container to a plateletpheresis        harness.    -   For the above stated uses, procedures should be developed and        records maintained, but licensees need not have FDA approval in        order to institute the procedures.

1. Using the STCD to Pool Blood Products

Appropriate use of an STCD to pool Platelets prepared from Whole Bloodcollection may obviate potential contamination from the spike and portentries commonly used. Pooling performed immediately before transfusionis an example of such appropriate use. Pooled Platelets should beadministered not more than 4 hours after pooling (See 21 CFR606.122(1)(2)).

However, pooling and subsequent storage may increase the risk comparedto administration of random donor units; if one contaminated unit ispooled with others and stored before administration, the total bacterialinoculum administered may be increased as a result of replication in theadditional volume. Accordingly, the proposed use of an STCD to pool andstore platelets for more than 4 hours should be supported by data whichsatisfactorily addresses whether such pooling is associated withincreased risk.

Such platelet pooling constitutes manufacture of a new product.

Pooling or mixing that involves platelets is considered the manufactureof a new product that requires submission and approval of a licenseapplication or application supplement if the storage period is to exceedfour hours.

2. Using the STCD to Prepare an Aliquot for Pediatric Use and DividedUnits

Pediatric units and divided units for Whole Blood, Red Blood Cells, andFresh Frozen Plasma prepared using an STCD will not be considered a newproduct for which a biologics license application (BLA) supplement isrequired providing the following conditions are met:

-   -   The manufacturer should have an approved biologics license or        license supplement, for the original (i.e., undivided) product,        including approval for each anticoagulant used.    -   Labels should be submitted for review and approval before        distribution. A notation should be made under the comments        section of FDA Form 2567, Transmittal of Labels and Circulars.    -   Final product containers approved for storage of the component        being prepared should be used.

Platelets manufactured under licensure must contain at least 5.5×(10)¹⁰platelets (21 CFR 640.24 (c)). Platelets, Pheresis manufactured underlicensure should contain at least 3.0×(10)¹¹ platelets (See RevisedGuideline for the Collection of Platelets, Pheresis, Oct. 7, 1988).

Procedures to be followed regarding the use of an STCD to preparedivided products from Whole Blood collections and from plasma andplatelets prepared by automated hemapheresis procedures should includedescriptions of:

-   -   How the apheresis harness or collection container will be        modified with an FDA-cleared STCD;    -   the minimum volume of the split plasma or whole blood products;    -   the volume and platelet concentration of the split        plateletpheresis products;    -   storage time of the product. The product should be in an        approved container and should be consistent with the storage        time on the label of such container;    -   method(s) to be used to label and track divided products in the        blood center's records.

NOTE: Procedures for labeling the aliquots should be clearly stated inthe procedure record keeping should be adequate to permit tracking andrecall of all components, if necessary.

3. Using an STCD to Connect Additional Saline or Anticoagulant LinesDuring an Automated Plasmapheresis Procedure

Procedures should be developed and records maintained consistent withthe instrument manufacturer's directions for use, but licensees need nothave FDA approval in order to institute the procedures.

4. Using the STCD to Attach Processing Solutions

When using an STCD to attach containers with processing solutions towashed or frozen red blood cell products, the dating period for theresulting products is 24 hours, unless data are provided in the form oflicense applications or application supplements to CBER to support alonger dating period (21 CFR 610.53(c)). Exemptions or modificationsmust be approved in writing from the Director, CBER (21 CFR 610.53(d)).

5. Using an STCD to Add an FDA-Cleared Leukocyte Reduction Filter

Some leuko-reduction filters are not integrally attached to the WholeBlood collection systems. Procedures for use of an STCD for pre-storagefiltration should be consistent with filter manufacturers' directionsfor use.

Leukocyte reduction prior to issue constitutes a major manufacturingchange. Therefore, for new leukocyte-reduced products prepared using anSTCD, manufacturers must submit biologics license applications (21 CFR601.2) or prior approval application supplements to FDA (21 CFR 601.12).

Using an STCD to remove samples from blood product containers fortesting (e.g., using an STCD to obtain a sample of platelets from acontainer of Platelets or Platelets, Pheresis for cross matching).

If the volume and/or cell count of the product after sample withdrawaldiffer from what is stated on the original label or in the circular ofinformation, the label on the product should be modified to reflect thenew volume and/or cell count. For example, samples may not be removedthat reduce the platelet count of a unit of Platelets to less than5.5×(10)¹⁰ platelets (21 CFR 640.24 (c)).

6. Additional Information from FDA Guidance

The FDA guidance presents general guidance as well as specificinformation and examples concerning specifications for submission ofapplications and application supplements to FDA addressing use of anSTCD. If further questions arise concerning appropriate use of an STCD,concerns should be directed to the Office of Blood Research and Review,Center for Biologics Evaluation and Research.

In some embodiments, the closed system uses one container from the timethe tumor fragments are obtained until the TILs are ready foradministration to the patient or cryopreserving. In some embodimentswhen two containers are used, the first container is a closedG-container and the population of TILs is centrifuged and transferred toan infusion bag without opening the first closed G-container. In someembodiments, when two containers are used, the infusion bag is aHypoThermosol-containing infusion bag. A closed system or closed TILcell culture system is characterized in that once the tumor sampleand/or tumor fragments have been added, the system is tightly sealedfrom the outside to form a closed environment free from the invasion ofbacteria, fungi, and/or any other microbial contamination.

In some embodiments, the reduction in microbial contamination is betweenabout 5% and about 100%. In some embodiments, the reduction in microbialcontamination is between about 5% and about 95%. In some embodiments,the reduction in microbial contamination is between about 5% and about90%. In some embodiments, the reduction in microbial contamination isbetween about 10% and about 90%. In some embodiments, the reduction inmicrobial contamination is between about 15% and about 85%. In someembodiments, the reduction in microbial contamination is about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%,about 99%, or about 100%.

The closed system allows for TIL growth in the absence and/or with asignificant reduction in microbial contamination.

Moreover, pH, carbon dioxide partial pressure and oxygen partialpressure of the TIL cell culture environment each vary as the cells arecultured. Consequently, even though a medium appropriate for cellculture is circulated, the closed environment still needs to beconstantly maintained as an optimal environment for TIL proliferation.To this end, it is desirable that the physical factors of pH, carbondioxide partial pressure and oxygen partial pressure within the cultureliquid of the closed environment be monitored by means of a sensor, thesignal whereof is used to control a gas exchanger installed at the inletof the culture environment, and the that gas partial pressure of theclosed environment be adjusted in real time according to changes in theculture liquid so as to optimize the cell culture environment. In someembodiments, the present invention provides a closed cell culture systemwhich incorporates at the inlet to the closed environment a gasexchanger equipped with a monitoring device which measures the pH,carbon dioxide partial pressure and oxygen partial pressure of theclosed environment, and optimizes the cell culture environment byautomatically adjusting gas concentrations based on signals from themonitoring device.

In some embodiments, the pressure within the closed environment iscontinuously or intermittently controlled. That is, the pressure in theclosed environment can be varied by means of a pressure maintenancedevice for example, thus ensuring that the space is suitable for growthof TILs in a positive pressure state, or promoting exudation of fluid ina negative pressure state and thus promoting cell proliferation. Byapplying negative pressure intermittently, moreover, it is possible touniformly and efficiently replace the circulating liquid in the closedenvironment by means of a temporary shrinkage in the volume of theclosed environment.

In some embodiments, optimal culture components for proliferation of theTILs can be substituted or added, and including factors such as IL-2and/or OKT3, as well as combination, can be added.

N. Cell Cultures

In an embodiment, a method for expanding TILs, including those discussabove as well as exemplified in FIG. 9 , may include using about 5,000mL to about 25,000 mL of cell medium, about 5,000 mL to about 10,000 mLof cell medium, or about 5,800 mL to about 8,700 mL of cell medium. Insome embodiments, the media is a serum free medium, as described forexample in Example 21. In some embodiments, the media in the firstexpansion is serum free. In some embodiments, the media in the secondexpansion is serum free. In some embodiments, the media in the firstexpansion and the second are both serum free. In an embodiment,expanding the number of TILs uses no more than one type of cell culturemedium. Any suitable cell culture medium may be used, e.g., AIM-V cellmedium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicinsulfate) cell culture medium (Invitrogen, Carlsbad Calif.). In thisregard, the inventive methods advantageously reduce the amount of mediumand the number of types of medium required to expand the number of TIL.In an embodiment, expanding the number of TIL may comprise feeding thecells no more frequently than every third or fourth day. Expanding thenumber of cells in a gas permeable container simplifies the proceduresnecessary to expand the number of cells by reducing the feedingfrequency necessary to expand the cells.

In an embodiment, the cell medium in the first and/or second gaspermeable container is unfiltered. The use of unfiltered cell medium maysimplify the procedures necessary to expand the number of cells. In anembodiment, the cell medium in the first and/or second gas permeablecontainer lacks beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TILs from the tumor tissue sample; expanding the number ofTILs in a second gas permeable container containing cell medium for aduration of about 7 to 14 days, e.g., about 11 days. In some embodimentspre-REP is about 7 to 14 days, e.g., about 11 days. In some embodiments,REP is about 7 to 14 days, e.g., about 11 days.

In an embodiment, TILs are expanded in gas-permeable containers.Gas-permeable containers have been used to expand TILs using PBMCs usingmethods, compositions, and devices known in the art, including thosedescribed in U.S. Patent Application Publication No. 2005/0106717 A1,the disclosures of which are incorporated herein by reference. In anembodiment, TILs are expanded in gas-permeable bags. In an embodiment,TILs are expanded using a cell expansion system that expands TILs in gaspermeable bags, such as the Xuri Cell Expansion System W25 (GEHealthcare). In an embodiment, TILs are expanded using a cell expansionsystem that expands TILs in gas permeable bags, such as the WAVEBioreactor System, also known as the Xuri Cell Expansion System W5 (GEHealthcare). In an embodiment, the cell expansion system includes a gaspermeable cell bag with a volume selected from the group consisting ofabout 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL,about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L,about 9 L, and about 10 L.

In an embodiment, TILs can be expanded in G-Rex flasks (commerciallyavailable from Wilson Wolf Manufacturing). Such embodiments allow forcell populations to expand from about 5×10⁵ cells/cm² to between 10×10⁶and 30×10⁶ cells/cm². In an embodiment this is without feeding. In anembodiment, this is without feeding so long as medium resides at aheight of about 10 cm in the G-Rex flask. In an embodiment this iswithout feeding but with the addition of one or more cytokines. In anembodiment, the cytokine can be added as a bolus without any need to mixthe cytokine with the medium. Such containers, devices, and methods areknown in the art and have been used to expand TILs, and include thosedescribed in U.S. Patent Application Publication No. US 2014/0377739A1,International Publication No. WO 2014/210036 A1, U.S. Patent ApplicationPublication No. us 2013/0115617 A1, International Publication No. WO2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228A1, U.S. Pat. No. 8,809,050 B2, International publication No. WO2011/072088 A2, U.S. Patent Application Publication No. US 2016/0208216A1, U.S. Patent Application Publication No. US 2012/0244133 A1,International Publication No. WO 2012/129201 A1, U.S. Patent ApplicationPublication No. US 2013/0102075 A1, U.S. Pat. No. 8,956,860 B2,International Publication No. WO 2013/173835 A1, U.S. Patent ApplicationPublication No. US 2015/0175966 A1, the disclosures of which areincorporated herein by reference. Such processes are also described inJin et al., J. Immunotherapy, 2012, 35:283-292.

O. Optional Genetic Engineering of TILs

In some embodiments, the TILs are optionally genetically engineered toinclude additional functionalities, including, but not limited to, ahigh-affinity T-cell receptor (TCR), e.g., a TCR targeted at atumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or achimeric antigen receptor (CAR) which binds to a tumor-associated cellsurface molecule (e.g., mesothelin) or lineage-restricted cell surfacemolecule (e.g., CD19).

P. Optional Cryopreservation of TILs

Either the bulk TIL population or the expanded population of TILs can beoptionally cryopreserved. In some embodiments, cryopreservation occurson the therapeutic TIL population. In some embodiments, cryopreservationoccurs on the TILs harvested after the second expansion. In someembodiments, cryopreservation occurs on the TILs in exemplary Step F ofFIG. 9 . In some embodiments, the TILs are cryopreserved in the infusionbag. In some embodiments, the TILs are cryopreserved prior to placementin an infusion bag. In some embodiments, the TILs are cryopreserved andnot placed in an infusion bag. In some embodiments, cryopreservation isperformed using a cryopreservation medium. In some embodiments, thecryopreservation media contains dimethylsulfoxide (DMSO). This isgenerally accomplished by putting the TIL population into a freezingsolution, e.g. 85% complement inactivated AB serum and 15% dimethylsulfoxide (DMSO). The cells in solution are placed into cryogenic vialsand stored for 24 hours at −80° C., with optional transfer to gaseousnitrogen freezers for cryopreservation. See, Sadeghi, et al., ActaOncologica 2013, 52, 978-986.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately ⅘ of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

In a preferred embodiment, a population of TILs is cryopreserved usingCS10 cryopreservation media (CryoStor 10, BioLife Solutions). In apreferred embodiment, a population of TILs is cryopreserved using acryopreservation media containing dimethylsulfoxide (DMSO). In apreferred embodiment, a population of TILs is cryopreserved using a 1:1(vol:vol) ratio of CS10 and cell culture media. In a preferredembodiment, a population of TILs is cryopreserved using about a 1:1(vol:vol) ratio of CS10 and cell culture media, further comprisingadditional IL-2.

As discussed above in Steps A through E, cryopreservation can occur atnumerous points throughout the TIL expansion process. In someembodiments, the bulk TIL population after the first expansion accordingto Step B or the expanded population of TILs after the one or moresecond expansions according to Step D can be cryopreserved.Cryopreservation can be generally accomplished by placing the TILpopulation into a freezing solution, e.g., 85% complement inactivated ABserum and 15% dimethyl sulfoxide (DMSO). The cells in solution areplaced into cryogenic vials and stored for 24 hours at −80° C., withoptional transfer to gaseous nitrogen freezers for cryopreservation. SeeSadeghi, et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately ⅘ of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

In some cases, the Step B TIL population can be cryopreservedimmediately, using the protocols discussed below. Alternatively, thebulk TIL population can be subjected to Step C and Step D and thencryopreserved after Step D. Similarly, in the case where geneticallymodified TILs will be used in therapy, the Step B or Step D TILpopulations can be subjected to genetic modifications for suitabletreatments.

Q. Optional Cell Viability Analyses

Optionally, a cell viability assay can be performed after the firstexpansion (sometimes referred to as the initial bulk expansion), usingstandard assays known in the art. For example, a trypan blue exclusionassay can be done on a sample of the bulk TILs, which selectively labelsdead cells and allows a viability assessment. Other assays for use intesting viability can include but are not limited to the Alamar blueassay; and the MTT assay.

1. Cell Counts, Viability, Flow Cytometry

In some embodiments, cell counts and/or viability are measured. Theexpression of markers such as but not limited CD3, CD4, CD8, and CD56,as well as any other disclosed or described herein, can be measured byflow cytometry with antibodies, for example but not limited to thosecommercially available from BD Bio-sciences (BD Biosciences, San Jose,Calif.) using a FACSCanto™ flow cytometer (BD Biosciences). The cellscan be counted manually using a disposable c-chip hemocytometer (VWR,Batavia, Ill.) and viability can be assessed using any method known inthe art, including but not limited to trypan blue staining.

In some cases, the bulk TIL population can be cryopreserved immediately,using the protocols discussed below. Alternatively, the bulk TILpopulation can be subjected to REP and then cryopreserved as discussedbelow. Similarly, in the case where genetically modified TILs will beused in therapy, the bulk or REP TIL populations can be subjected togenetic modifications for suitable treatments.

2. Cell Cultures

In an embodiment, a method for expanding TILs may include using about5,000 mL to about 25,000 mL of cell medium, about 5,000 mL to about10,000 mL of cell medium, or about 5,800 mL to about 8,700 mL of cellmedium. In an embodiment, expanding the number of TILs uses no more thanone type of cell culture medium. Any suitable cell culture medium may beused, e.g., AIM-V cell medium (L-glutamine, 50 μM streptomycin sulfate,and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, CarlsbadCalif.). In this regard, the inventive methods advantageously reduce theamount of medium and the number of types of medium required to expandthe number of TIL. In an embodiment, expanding the number of TIL maycomprise feeding the cells no more frequently than every third or fourthday. Expanding the number of cells in a gas permeable containersimplifies the procedures necessary to expand the number of cells byreducing the feeding frequency necessary to expand the cells.

In an embodiment, the cell medium in the first and/or second gaspermeable container is unfiltered. The use of unfiltered cell medium maysimplify the procedures necessary to expand the number of cells. In anembodiment, the cell medium in the first and/or second gas permeablecontainer lacks beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TILs from the tumor tissue sample; expanding the number ofTILs in a second gas permeable container containing cell medium thereinusing aAPCs for a duration of about 14 to about 42 days, e.g., about 28days.

In an embodiment, TILs are expanded in gas-permeable containers.Gas-permeable containers have been used to expand TILs using PBMCs usingmethods, compositions, and devices known in the art, including thosedescribed in U.S. Patent Application Publication No. 2005/0106717 A1,the disclosures of which are incorporated herein by reference. In anembodiment, TILs are expanded in gas-permeable bags. In an embodiment,TILs are expanded using a cell expansion system that expands TILs in gaspermeable bags, such as the Xuri Cell Expansion System W25 (GEHealthcare). In an embodiment, TILs are expanded using a cell expansionsystem that expands TILs in gas permeable bags, such as the WAVEBioreactor System, also known as the Xuri Cell Expansion System W5 (GEHealthcare). In an embodiment, the cell expansion system includes a gaspermeable cell bag with a volume selected from the group consisting ofabout 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL,about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L,about 9 L, and about 10 L.

In an embodiment, TILs can be expanded in G-Rex flasks (commerciallyavailable from Wilson Wolf Manufacturing). Such embodiments allow forcell populations to expand from about 5×10⁵ cells/cm² to between 10×10⁶and 30×10⁶ cells/cm². In an embodiment this is without feeding. In anembodiment, this is without feeding so long as medium resides at aheight of about 10 cm in the G-Rex flask. In an embodiment this iswithout feeding but with the addition of one or more cytokines. In anembodiment, the cytokine can be added as a bolus without any need to mixthe cytokine with the medium. Such containers, devices, and methods areknown in the art and have been used to expand TILs, and include thosedescribed in U.S. Patent Application Publication No. US 2014/0377739A1,International Publication No. WO 2014/210036 A1, U.S. Patent ApplicationPublication No. us 2013/0115617 A1, International Publication No. WO2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228A1, U.S. Pat. No. 8,809,050 B2, International publication No. WO2011/072088 A2, U.S. Patent Application Publication No. US 2016/0208216A1, U.S. Patent Application Publication No. US 2012/0244133 A1,International Publication No. WO 2012/129201 A1, U.S. Patent ApplicationPublication No. US 2013/0102075 A1, U.S. Pat. No. 8,956,860 B2,International Publication No. WO 2013/173835 A1, U.S. Patent ApplicationPublication No. US 2015/0175966 A1, the disclosures of which areincorporated herein by reference. Such processes are also described inJin et al., J. Immunotherapy, 2012, 35:283-292. Optional GeneticEngineering of TILs

In some embodiments, the TILs are optionally genetically engineered toinclude additional functionalities, including, but not limited to, ahigh-affinity T-cell receptor (TCR), e.g., a TCR targeted at atumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or achimeric antigen receptor (CAR) which binds to a tumor-associated cellsurface molecule (e.g., mesothelin) or lineage-restricted cell surfacemolecule (e.g., CD19).

IV. Methods of Treating Patients

Methods of treatment begin with the initial TIL collection and cultureof TILs. Such methods have been both described in the art by, forexample, Jin et al., J. Immunotherapy, 2012, 35(3):283-292, incorporatedby reference herein in its entirety. Embodiments of methods of treatmentare described throughout the sections below, including the Examples.

The expanded TILs produced according the methods described herein,including, for example as described in Steps A through F above oraccording to Steps A through F above (also as shown, for example, inFIG. 2A and/or FIG. 9 find particular use in the treatment of patientswith cancer (for example, as described in Goff, et al., J. ClinicalOncology, 2016, 34(20):2389-239, as well as the supplemental content;incorporated by reference herein in its entirety. In some embodiments,TIL were grown from resected deposits of metastatic melanoma aspreviously described (see, Dudley, et al., J Immunother., 2003,26:332-342; incorporated by reference herein in its entirety). Freshtumor can be dissected under sterile conditions. A representative samplecan be collected for formal pathologic analysis. Single fragments of 2mm³ to 3 mm³ may be used. In some embodiments, 5, 10, 15, 20, 25 or 30samples per patient are obtained. In some embodiments, 20, 25, or 30samples per patient are obtained. In some embodiments, 20, 22, 24, 26,or 28 samples per patient are obtained. In some embodiments, 24 samplesper patient are obtained. Samples can be placed in individual wells of a24-well plate, maintained in growth media with high-dose IL-2 (6,000IU/mL), and monitored for destruction of tumor and/or proliferation ofTIL. Any tumor with viable cells remaining after processing can beenzymatically digested into a single cell suspension and cryopreserved,as described herein.

In some embodiments, successfully grown TIL can be sampled for phenotypeanalysis (CD3, CD4, CD8, and CD56) and tested against autologous tumorwhen available. TIL can be considered reactive if overnight cocultureyielded interferon-gamma (IFN-γ) levels >200 pg/mL and twice background.(Goff, et al., J Immunother., 2010, 33:840-847; incorporated byreference herein in its entirety). In some embodiments, cultures withevidence of autologous reactivity or sufficient growth patterns can beselected for a second expansion (for example, a second expansion asprovided in according to Step D of FIG. 2A and/or FIG. 9 , includingsecond expansions that are sometimes referred to as rapid expansion(REP). In some embodiments, expanded TILs with high autologousreactivity (for example, high proliferation during a second expansion),are selected for an additional second expansion. In some embodiments,TILs with high autologous reactivity (for example, high proliferationduring second expansion as provided in Step D of FIG. 2A and/or FIG. 9 ,are selected for an additional second expansion according to Step D ofFIG. 2A and/or FIG. 9 .

In some embodiments, the patient is not moved directly to ACT (adoptivecell transfer), for example, in some embodiments, after tumor harvestingand/or a first expansion, the cells are not utilized immediately. Insome embodiments, TILs can be cryopreserved and thawed 2 days beforeadministration to a patient. In some embodiments, TILs can becryopreserved and thawed 1 day before administration to a patient. Insome embodiments, the TILs can be cryopreserved and thawed immediatelybefore the administration to a patient.

Cell phenotypes of cryopreserved samples of infusion bag TIL can beanalyzed by flow cytometry (e.g., FlowJo) for surface markers CD3, CD4,CD8, CCR7, and CD45RA (BD BioSciences), as well as by any of the methodsdescribed herein. Serum cytokines can be measured by using standardenzyme-linked immunosorbent assay techniques. A rise in serum IFN-γ canbe defined as >100 pg/mL and at least 4-fold or at least 3-fold or atleast 2-fold or at least 1-fold greater than baseline levels of serumIFN-γ. In some embodiments, a rise in serum IFN-γ is defined as >1000pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >200pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >250pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >300pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >350pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >400pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >450pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >500pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >550pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >600pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >650pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >700pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >750pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >800pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >850pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >900pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >950pg/mL. In some embodiments, a rise in serum IFN-γ is defined as >1000pg/mL.

In some embodiments, the TILs produced by the methods provided herein,for example those exemplified in FIG. 2A and/or FIG. 9 , provide for asurprising improvement in clinical efficacy of the TILs. In someembodiments, the TILs produced by the methods provided herein, forexample those exemplified in FIG. 2A and/or FIG. 9 , exhibit increasedclinical efficacy as compared to TILs produced by methods other thanthose described herein, including, for example, methods other than thoseexemplified in FIG. 2A and/or FIG. 9 . In some embodiments, the methodsother than those described herein include methods referred to as process1C and/or Generation 1 (Gen 1). In some embodiments, the increasedefficacy is measured by DCR, ORR, and/or other clinical responses. Insome embodiments, the TILs produced by the methods provided herein, forexample those exemplified in FIG. 2A and/or FIG. 9 , exhibit a similartime to response and safety profile compared to TILs produced by methodsother than those described herein, including, for example, methods otherthan those exemplified in FIG. 2A and/or FIG. 9 , for example the Gen 1process.

In some embodiments, IFN-gamma (IFN-γ) is indicative of treatmentefficacy and/or increased clinical efficacy. In some embodiments, IFN-γin the blood of subjects treated with TILs is indicative of active TILs.In some embodiments, a potency assay for IFN-γ production is employed.IFN-γ production is another measure of cytotoxic potential. IFN-γproduction can be measured by determining the levels of the cytokineIFN-γ in the blood, serum, or TILs ex vivo of a subject treated withTILs prepared by the methods of the present invention, including thoseas described for example in FIG. 2A and/or FIG. 9 . In some embodiments,an increase in IFN-γ is indicative of treatment efficacy in a patienttreated with the TILs produced by the methods of the present invention.In some embodiments, IFN-γ is increased one-fold, two-fold, three-fold,four-fold, or five-fold or more as compared to an untreated patientand/or as compared to a patient treated with TILs prepared using othermethods than those provide herein including, for example, methods otherthan those embodied in FIG. 2A and/or FIG. 9 . In some embodiments,IFN-γ secretion is increased one-fold as compared to an untreatedpatient and/or as compared to a patient treated with TILs prepared usingother methods than those provided herein including, for example, methodsother than those embodied in FIG. 2A and/or FIG. 9 . In someembodiments, IFN-γ secretion is increased two-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including, forexample, methods other than those embodied in FIG. 2A and/or FIG. 9 . Insome embodiments, IFN-γ secretion is increased three-fold as compared toan untreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including, forexample, methods other than those embodied in FIG. 2A and/or FIG. 9 . Insome embodiments, IFN-γ secretion is increased four-fold as compared toan untreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including, forexample, methods other than those embodied in FIG. 2A and/or FIG. 9 . Insome embodiments, IFN-γ secretion is increased five-fold as compared toan untreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including, forexample, methods other than those embodied in FIG. 2A and/or FIG. 9 . Insome embodiments, IFN-γ is measured using a Quantikine ELISA kit. Insome embodiments, IFN-γ is measured in TILs ex vivo of a subject treatedwith TILs prepared by the methods of the present invention, includingthose as described for example in FIG. 2A and/or FIG. 9 . In someembodiments, IFN-γ is measured in blood of a subject treated with TILsprepared by the methods of the present invention, including those asdescribed for example in FIG. 2A and/or FIG. 9 . In some embodiments,IFN-γ is measured in TILs serum of a subject treated with TILs preparedby the methods of the present invention, including those as describedfor example in FIG. 2A and/or FIG. 9 .

In some embodiments, the TILs prepared by the methods of the presentinvention, including those as described for example in FIG. 2A and/orFIG. 9 , exhibit increased polyclonality as compared to TILs produced byother methods, including those not exemplified in FIG. 2A and/or FIG. 9, such as for example, methods referred to as process 1C methods. Insome embodiments, significantly improved polyclonality and/or increasedpolyclonality is indicative of treatment efficacy and/or increasedclinical efficacy. In some embodiments, polyclonality refers to theT-cell repertoire diversity. In some embodiments, an increase inpolyclonality can be indicative of treatment efficacy with regard toadministration of the TILs produced by the methods of the presentinvention. In some embodiments, polyclonality is increased one-fold,two-fold, ten-fold, 100-fold, 500-fold, or 1000-fold as compared to TILsprepared using methods than those provide herein including, for example,methods other than those embodied in FIG. 2A and/or FIG. 9 . In someembodiments, polyclonality is increased one-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including, forexample, methods other than those embodied in FIG. 2A and/or FIG. 9 . Insome embodiments, polyclonality is increased two-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including, forexample, methods other than those embodied in FIG. 2A and/or FIG. 9 . Insome embodiments, polyclonality is increased ten-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including, forexample, methods other than those embodied in FIG. 2A and/or FIG. 9 . Insome embodiments, polyclonality is increased 100-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including, forexample, methods other than those embodied in FIG. 2A and/or FIG. 9 . Insome embodiments, polyclonality is increased 500-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including, forexample, methods other than those embodied in FIG. 2A and/or FIG. 9 . Insome embodiments, polyclonality is increased 1000-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including, forexample, methods other than those embodied in FIG. 2A and/or FIG. 9 .

Measures of efficacy can include the disease control rate (DCR) as wellas overall response rate (ORR), as known in the art as well as describedherein.

A. Methods of Treating Cancers

The compositions and methods described herein can be used in a methodfor treating diseases. In an embodiment, they are for use in treatinghyperproliferative disorders, such as cancer, in an adult patient or ina pediatric patient. They may also be used in treating other disordersas described herein and in the following paragraphs.

In some embodiments, the hyperproliferative disorder is cancer. In someembodiments, the hyperproliferative disorder is a solid tumor cancer. Insome embodiments, the solid tumor cancer is selected from the groupconsisting of anal cancer, bladder cancer, breast cancer (includingtriple-negative breast cancer), bone cancer, cancer caused by humanpapilloma virus (HPV), central nervous system associated cancer(including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma,and primitive neuroectodermal tumor), cervical cancer (includingsquamous cell cervical cancer, adenosquamous cervical cancer, andcervical adenocarcinoma), colon cancer, colorectal cancer, endometrialcancer, esophageal cancer, esophagogastric junction cancer, gastriccancer, gastrointestinal cancer, gastrointestinal stromal tumor,glioblastoma, glioma, head and neck cancer (including head and necksquamous cell carcinoma (HNSCC), hypopharynx cancer, larynx cancer,nasopharynx cancer, oropharynx cancer, and pharynx cancer), kidneycancer, liver cancer, lung cancer (including non-small-cell lung cancer(NSCLC) and small-cell lung cancer), melanoma (including uveal melanoma,choroidal melanoma, ciliary body melanoma, or iris melanoma),mesothelioma (including malignant pleural mesothelioma), ovarian cancer,pancreatic cancer (including pancreatic ductal adenocarcinoma), penilecancer, rectal cancer, renal cancer, renal cell carcinoma, sarcoma(including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other boneand soft tissue sarcomas), thyroid cancer (including anaplastic thyroidcancer), uterine cancer, and vaginal cancer.

In some embodiments, the hyperproliferative disorder is a hematologicalmalignancy. In some embodiments, the hematological malignancy isselected from the group consisting of chronic lymphocytic leukemia,acute lymphoblastic leukemia, diffuse large B cell lymphoma,non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, mantlecell lymphoma, and multiple myeloma. In some embodiments, the presentinvention includes a method of treating a patient with a cancer, whereinthe cancer is a hematological malignancy. In some embodiments, thepresent invention includes a method of treating a patient with a cancerusing TILs, MILs, or PBLs modified to express one or more CCRs, whereinthe cancer is a hematological malignancy. In some embodiments, thepresent invention includes a method of treating a patient with a cancerusing MILs or PBLs modified to express one or more CCRs, wherein thecancer is a hematological malignancy.

In an embodiment, the cancer is one of the foregoing cancers, includingsolid tumor cancers and hematological malignancies, that is relapsed orrefractory to treatment with at least one prior therapy, includingchemotherapy, radiation therapy, or immunotherapy. In an embodiment, thecancer is one of the foregoing cancers that is relapsed or refractory totreatment with at least two prior therapies, including chemotherapy,radiation therapy, and/or immunotherapy. In an embodiment, the cancer isone of the foregoing cancers that is relapsed or refractory to treatmentwith at least three prior therapies, including chemotherapy, radiationtherapy, and/or immunotherapy.

In some embodiments, the cancer is a microsatellite instability-high(MSI-H) or a mismatch repair deficient (dMMR) cancer. MSI-H and dMMRcancers and testing therefore have been described in Kawakami, et al.,Curr. Treat. Options Oncol. 2015, 16, 30, the disclosures of which areincorporated by reference herein.

In some embodiments, the present invention includes a method of treatinga patient with a cancer using TILs, MILs, or PBLs modified to expressone or more CCRs, wherein the patient is a human. In some embodiments,the present invention includes a method of treating a patient with acancer using TILs, MILs, or PBLs modified to express one or more CCRs,wherein the patient is a non-human. In some embodiments, the presentinvention includes a method of treating a patient with a cancer usingTILs, MILs, or PBLs modified to express one or more CCRs, wherein thepatient is a companion animal. In some embodiments, the presentinvention includes a method of treating a patient with a cancer usingTILs, MILs, or PBLs modified to express one or more CCRs, wherein thepatient is a primate, equine, canine, or feline animal.

In some embodiments, the present invention includes a method of treatinga patient with a cancer, wherein the cancer is refractory to treatmentwith a BRAF inhibitor and/or a MEK inhibitor. In some embodiments, thepresent invention includes a method of treating a patient with a cancer,wherein the cancer is refractory to treatment with a BRAF inhibitorselected from the group consisting of vemurafenib, dabrafenib,encorafenib, sorafenib, and pharmaceutically acceptable salts orsolvates thereof. In some embodiments, the present invention includes amethod of treating a patient with a cancer, wherein the cancer isrefractory to treatment with a MEK inhibitor selected from the groupconsisting of trametinib, cobimetinib, binimetinib, selumetinib,pimasertinib, refametinib, and pharmaceutically acceptable salts orsolvates thereof. In some embodiments, the present invention includes amethod of treating a patient with a cancer, wherein the cancer isrefractory to treatment with a BRAF inhibitor selected from the groupconsisting of vemurafenib, dabrafenib, encorafenib, sorafenib, andpharmaceutically acceptable salts or solvates thereof, and a MEKinhibitor selected from the group consisting of trametinib, cobimetinib,binimetinib, selumetinib, pimasertinib, refametinib, andpharmaceutically acceptable salts or solvates thereof.

In some embodiments, the present invention includes a method of treatinga patient with a cancer, wherein the cancer is a pediatric cancer.

In some embodiments, the present invention includes a method of treatinga patient with a cancer wherein the cancer is uveal melanoma.

In some embodiments, the present invention includes a method of treatinga patient with a cancer, wherein the uveal melanoma is choroidalmelanoma, ciliary body melanoma, or iris melanoma.

In some embodiments, the present invention includes a method of treatinga patient with a cancer, wherein the pediatric cancer is aneuroblastoma.

In some embodiments, the present invention includes a method of treatinga patient with a cancer, wherein the pediatric cancer is a sarcoma.

In some embodiments, the present invention includes a method of treatinga patient with a cancer, wherein the sarcoma is osteosarcoma.

In some embodiments, the present invention includes a method of treatinga patient with a cancer, wherein the sarcoma is a soft tissue sarcoma.

In some embodiments, the present invention includes a method of treatinga patient with a cancer, wherein the soft tissue sarcoma isrhabdomyosarcoma, Ewing sarcoma, or primitive neuroectodermal tumor(PNET).

In some embodiments, the present invention includes a method of treatinga patient with a cancer, wherein the pediatric cancer is a centralnervous system (CNS) associated cancer. In some embodiments, thepediatric cancer is refractory to treatment with chemotherapy. In someembodiments, the pediatric cancer is refractory to treatment withradiation therapy. In some embodiments, the pediatric cancer isrefractory to treatment with dinutuximab.

In some embodiments, the present invention includes a method of treatinga patient with a cancer, wherein the CNS associated cancer ismedulloblastoma, pineoblastoma, glioma, ependymoma, or glioblastoma.

The compositions and methods described herein can be used in a methodfor treating cancer, wherein the cancer is refractory or resistant toprior treatment with an anti-PD-1 or anti-PD-L1 antibody. In someembodiments, the patient is a primary refractory patient to an anti-PD-1or anti-PD-L1 antibody. In some embodiments, the patient shows no priorresponse to an anti-PD-1 or anti-PD-L1 antibody. In some embodiments,the patient shows a prior response to an anti-PD-1 or anti-PD-L1antibody, follow by progression of the patient's cancer. In someembodiments, the cancer is refractory to an anti-CTLA-4 antibody and/oran anti-PD-1 or anti-PD-L1 antibody in combination with at least onechemotherapeutic agent. In some embodiments, the prior chemotherapeuticagent is carboplatin, paclitaxel, pemetrexed, and/or cisplatin. In someprior embodiments, the chemotherapeutic agent(s) is a platinum doubletchemotherapeutic agent. In some embodiments, the platinum doublettherapy comprises a first chemotherapeutic agent selected from the groupconsisting of cisplatin and carboplatin and a second chemotherapeuticagent selected from the group consisting of vinorelbine, gemcitabine anda taxane (including for example, paclitaxel, docetaxel ornab-paclitaxel). In some embodiments, the platinum doubletchemotherapeutic agent is in combination with pemetrexed.

In some embodiments, the NSCLC is PD-L1 negative and/or is from apatient with a cancer that expresses PD-L1 with a tumor proportion score(TPS) of <1%, as described elsewhere herein.

In some embodiments, the NSCLC is refractory to a combination therapycomprising an anti-PD-1 or the anti-PD-L1 antibody and a platinumdoublet therapy, wherein the platinum doublet therapy comprises:

-   -   i) a first chemotherapeutic agent selected from the group        consisting of cisplatin and carboplatin,    -   ii) and a second chemotherapeutic agent selected from the group        consisting of vinorelbine, gemcitabine and a taxane (including        for example, paclitaxel, docetaxel or nab-paclitaxel).

In some embodiments, the NSCLC is refractory to a combination therapycomprising an anti-PD-1 or the anti-PD-L1 antibody, pemetrexed, and aplatinum doublet therapy, wherein the platinum doublet therapycomprises:

-   -   i) a first chemotherapeutic agent selected from the group        consisting of cisplatin and carboplatin,    -   ii) and a second chemotherapeutic agent selected from the group        consisting of vinorelbine, gemcitabine and a taxane (including        for example, paclitaxel, docetaxel or nab-paclitaxel).

In some embodiments, the NSCLC has been treated with an anti-PD-1antibody. In some embodiments, the NSCLC has been treated with ananti-PD-L1 antibody. In some embodiments, the NSCLC patient is treatmentnaïve. In some embodiments, the NSCLC has not been treated with ananti-PD-1 antibody. In some embodiments, the NSCLC has not been treatedwith an anti-PD-L1 antibody. In some embodiments, the NSCLC has beenpreviously treated with a chemotherapeutic agent. In some embodiments,the NSCLC has been previously treated with a chemotherapeutic agent butis not longer being treated with the chemotherapeutic agent. In someembodiments, the NSCLC patient is anti-PD-1/PD-L1 naïve. In someembodiments, the NSCLC patient has low expression of PD-L1. In someembodiments, the NSCLC patient has treatment naïve NSCLC or ispost-chemotherapeutic treatment but anti-PD-1/PD-L1 naïve. In someembodiments, the NSCLC patient is treatment naïve orpost-chemotherapeutic treatment but anti-PD-1/PD-L1 naïve and has lowexpression of PD-L1. In some embodiments, the NSCLC patient has bulkydisease at baseline. In some embodiments, the subject has bulky diseaseat baseline and has low expression of PD-L1. In some embodiments, theNSCLC patient has no detectable expression of PD-L1. In someembodiments, the NSCLC patient is treatment naïve orpost-chemotherapeutic treatment but anti-PD-1/PD-L1 naïve and has nodetectable expression of PD-L1. In some embodiments, the patient hasbulky disease at baseline and has no detectable expression of PD-L1. Insome embodiments, the NSCLC patient has treatment naïve NSCLC or postchemotherapy (e.g., post chemotherapeutic agent) but anti-PD-1/PD-L1naïve who have low expression of PD-L1 and/or have bulky disease atbaseline. In some embodiments, bulky disease is indicated where themaximal tumor diameter is greater than 7 cm measured in either thetransverse or coronal plane. In some embodiments, bulky disease isindicated when there are swollen lymph nodes with a short-axis diameterof 20 mm or greater. In some embodiments, the chemotherapeutic includesa standard of care therapeutic for NSCLC.

In some embodiments, PD-L1 expression is determined by the tumorproportion score. In some embodiments, the subject with a refractoryNSCLC tumor has a <1% tumor proportion score (TPS). In some embodiments,the subject with a refractory NSCLC tumor has a >1% TPS. In someembodiments, subject with the refractory NSCLC has been previouslytreated with an anti-PD-1 and/or anti-PD-L1 antibody and the tumorproportion score was determined prior to said anti-PD-1 and/oranti-PD-L1 antibody treatment. In some embodiments, subject with therefractory NSCLC has been previously treated with an anti-PD-L1 antibodyand the tumor proportion score was determined prior to said anti-PD-L1antibody treatment.

In some embodiments, the TILs prepared by the methods of the presentinvention, including those as described for example in FIG. 1 , exhibitincreased polyclonality as compared to TILs produced by other methods,including those not exemplified in FIG. 1 , such as for example, methodsreferred to as process 1C methods. In some embodiments, significantlyimproved polyclonality and/or increased polyclonality is indicative oftreatment efficacy and/or increased clinical efficacy for cancertreatment. In some embodiments, polyclonality refers to the T-cellrepertoire diversity. In some embodiments, an increase in polyclonalitycan be indicative of treatment efficacy with regard to administration ofthe TILs produced by the methods of the present invention. In someembodiments, polyclonality is increased one-fold, two-fold, ten-fold,100-fold, 500-fold, or 1000-fold as compared to TILs prepared usingmethods than those provide herein including for example, methods otherthan those embodied in FIG. 1 . In some embodiments, polyclonality isincreased one-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 1 . In some embodiments, polyclonality isincreased two-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 1 . In some embodiments, polyclonality isincreased ten-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 1 . In some embodiments, polyclonality isincreased 100-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 1 . In some embodiments, polyclonality isincreased 500-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 1 . In some embodiments, polyclonality isincreased 1000-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 1 .

In some embodiments, PD-L1 expression is determined by the tumorproportion score using one more testing methods as described herein. Insome embodiments, the subject or patient with a NSCLC tumor has a <1%tumor proportion score (TPS). In some embodiments, the NSCLC tumor hasa >1% TPS. In some embodiments, the subject or patient with the NSCLChas been previously treated with an anti-PD-1 and/or anti-PD-L1 antibodyand the tumor proportion score was determined prior to the anti-PD-1and/or anti-PD-L1 antibody treatment. In some embodiments, the subjector patient with the NSCLC has been previously treated with an anti-PD-L1antibody and the tumor proportion score was determined prior to theanti-PD-L1 antibody treatment. In some embodiments, the subject orpatient with a refractory or resistant NSCLC tumor has a <1% tumorproportion score (TPS). In some embodiments, the subject or patient witha refractory or resistant NSCLC tumor has a >1% TPS. In someembodiments, the subject or patient with the refractory or resistantNSCLC has been previously treated with an anti-PD-1 and/or anti-PD-L1antibody and the tumor proportion score was determined prior to theanti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, thesubject or patient with the refractory or resistant NSCLC has beenpreviously treated with an anti-PD-L1 antibody and the tumor proportionscore was determined prior to the anti-PD-L1 antibody treatment.

In some embodiments, the NSCLC is an NSCLC that exhibits a tumorproportion score (TPS), or the percentage of viable tumor cells from apatient taken prior to anti-PD-1 or anti-PD-L1 therapy, showing partialor complete membrane staining at any intensity, for the PD-L1 proteinthat is less than 1% (TPS <1%). In an embodiment, the NSCLC is an NSCLCthat exhibits a TPS selected from the group consisting of <50%, <45%,<40%, <35%, <30%, <25%, <20%, <15%, <10%, <9%, <8%, <7%, <6%, <5%, <4%,<3%, <2%, <1%, <0.9%, <0.8%, <0.7%, <0.6%, <0.5%, <0.4%, <0.3%, <0.2%,<0.1%, <0.09%, <0.08%, <0.07%, <0.06%, <0.05%, <0.04%, <0.03%, <0.02%,and <0.01%. In an embodiment, the NSCLC is an NSCLC that exhibits a TPSselected from the group consisting of about 50%, about 45%, about 40%,about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09%, about0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%,about 0.02%, and about 0.01%. In an embodiment, the NSCLC is an NSCLCthat exhibits a TPS between 0% and 1%. In an embodiment, the NSCLC is anNSCLC that exhibits a TPS between 0% and 0.9%. In an embodiment, theNSCLC is an NSCLC that exhibits a TPS between 0% and 0.8%. In anembodiment, the NSCLC is an NSCLC that exhibits a TPS between 0% and0.7%. In an embodiment, the NSCLC is an NSCLC that exhibits a TPSbetween 0% and 0.6%. In an embodiment, the NSCLC is an NSCLC thatexhibits a TPS between 0% and 0.5%. In an embodiment, the NSCLC is anNSCLC that exhibits a TPS between 0% and 0.4%. In an embodiment, theNSCLC is an NSCLC that exhibits a TPS between 0% and 0.3%. In anembodiment, the NSCLC is an NSCLC that exhibits a TPS between 0% and0.2%. In an embodiment, the NSCLC is an NSCLC that exhibits a TPSbetween 0% and 0.1%. TPS may be measured by methods known in the art,such as those described in Hirsch, et al. J. Thorac. Oncol. 2017, 12,208-222 or those used for the determination of TPS prior to treatmentwith pembrolizumab or other anti-PD-1 or anti-PD-L1 therapies. Methodsfor measurement of TPS that have been approved by the U.S. Food and DrugAdministration may also be used. In some embodiments, the PD-L1 isexosomal PD-L1. In some embodiments, the PD-L1 is found on circulatingtumor cells.

In some embodiments, the partial membrane staining includes 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 99%, or more. In some embodiments, the completedmembrane staining includes approximately 100% membrane staining.

In some embodiments, testing for PD-L1 can involve measuring levels ofPD-L1 in patient serum. In these embodiments, measurement of PD-L1 inpatient serum removes the uncertainty of tumor heterogeneity and thepatient discomfort of serial biopsies.

In some embodiments, elevated soluble PD-L1 as compared to a baseline orstandard level correlates with worsened prognosis in NSCLC. See, forexample, Okuma, et al., Clinical Lung Cancer, 2018, 19, 410-417;Vecchiarelli, et al., Oncotarget, 2018, 9, 17554-17563. In someembodiments, the PD-L1 is exosomal PD-L1. In some embodiments, the PD-L1is expressed on circulating tumor cells.

In an embodiment, the invention provides a method of treating non-smallcell lung carcinoma (NSCLC) by administering a population of tumorinfiltrating lymphocytes (TILs) to a subject or patient in need thereof,wherein the subject or patient has at least one of:

a predetermined tumor proportion score (TPS) of PD-L1<1%,

a TPS score of PD-L1 of 1%-49%, or

a predetermined absence of one or more driver mutations,

wherein the driver mutation is selected from the group consisting of anEGFR mutation, an EGFR insertion, an EGFR exon 20 mutation, a KRASmutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1mutation), a ROS1 fusion, a RET mutation, a RET fusion, an ERBB2mutation, an ERBB2 amplification, a BRCA mutation, a MAP2K1 mutation,PIK3CA, CDKN2A, a PTEN mutation, an UMD mutation, an NRAS mutation, aKRAS mutation, an NF1 mutation, a MET mutation, a MET splice and/oraltered MET signaling, a TP53 mutation, a CREBBP mutation, a KMT2Cmutation, a KMT2D mutation, an ARID1A mutation, a RB1 mutation, an ATMmutation, a SETD2 mutation, a FLT3 mutation, a PTPN11 mutation, a FGFR1mutation, an EP300 mutation, a MYC mutation, an EZH2 mutation, a JAK2mutation, a FBXW7 mutation, a CCND3 mutation, and a GNA11 mutation, andwherein the method comprises:

(a) obtaining and/or receiving a first population of TILs from a tumorresected from the subject or patient by processing a tumor sampleobtained from the subject into multiple tumor fragments;

(b) adding the first population of TILs into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 to produce a secondpopulation of TILs, wherein the first expansion is performed in a closedcontainer providing a first gas-permeable surface area, wherein thefirst expansion is performed for about 3-14 days to obtain the secondpopulation of TILs, wherein the second population of TILs is at least50-fold greater in number than the first population of TILs, and whereinthe transition from step (b) to step (c) occurs without opening thesystem;

(d) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, OKT-3, andantigen presenting cells (APCs), to produce a third population of TILs,wherein the second expansion is performed for about 7-14 days to obtainthe third population of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (c) to step (d) occurswithout opening the system;

(e) harvesting therapeutic population of TILs obtained from step (d),wherein the transition from step (d) to step (e) occurs without openingthe system; and

(f) transferring the harvested TIL population from step (e) to aninfusion bag, wherein the transfer from step (e) to (f) occurs withoutopening the system;

(g) cryopreserving the infusion bag comprising the harvested TILpopulation from step (f) using a cryopreservation process; and

(h) administering a therapeutically effective dosage of the thirdpopulation of TILs from the infusion bag in step (g) to the subject orpatient.

In an embodiment, the invention provides a method of treating non-smallcell lung carcinoma (NSCLC) by administering a population of tumorinfiltrating lymphocytes (TILs) to a patient in need thereof, whereinthe method comprises:

(a) testing the patient's tumor for PD-L1 expression and tumorproportion score (TPS) of PD-L1,

(b) testing the patient for the absence of one or more driver mutations,wherein the driver mutation is selected from the group consisting of anEGFR mutation, an EGFR insertion, an EGFR exon 20 mutation, a KRASmutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1mutation), a ROS1 fusion, a RET mutation, a RET fusion, an ERBB2mutation, an ERBB2 amplification, a BRCA mutation, a MAP2K1 mutation,PIK3CA, CDKN2A, a PTEN mutation, an UMD mutation, an NRAS mutation, aKRAS mutation, an NF1 mutation, a MET mutation, a MET splice and/oraltered MET signaling, a TP53 mutation, a CREBBP mutation, a KMT2Cmutation, a KMT2D mutation, an ARID1A mutation, a RB1 mutation, an ATMmutation, a SETD2 mutation, a FLT3 mutation, a PTPN11 mutation, a FGFR1mutation, an EP300 mutation, a MYC mutation, an EZH2 mutation, a JAK2mutation, a FBXW7 mutation, a CCND3 mutation, and a GNA11 mutation,

(c) determining that the patient has a TPS score for PD-L1 of about 1%to about 49% and determining that the patient also has no drivermutations,

(d) obtaining and/or receiving a first population of TILs from a tumorresected from the subject or patient by processing a tumor sampleobtained from the subject into multiple tumor fragments;

(e) adding the first population of TILs into a closed system;

(f) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 to produce a secondpopulation of TILs, wherein the first expansion is performed in a closedcontainer providing a first gas-permeable surface area, wherein thefirst expansion is performed for about 3-14 days to obtain the secondpopulation of TILs, wherein the second population of TILs is at least50-fold greater in number than the first population of TILs, and whereinthe transition from step (e) to step (f) occurs without opening thesystem;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, OKT-3, andantigen presenting cells (APCs), to produce a third population of TILs,wherein the second expansion is performed for about 7-14 days to obtainthe third population of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (f) to step (g) occurswithout opening the system;

(h) harvesting therapeutic population of TILs obtained from step (d),wherein the transition from step (d) to step (e) occurs without openingthe system; and

(i) transferring the harvested TIL population from step (e) to aninfusion bag, wherein the transfer from step (e) to (f) occurs withoutopening the system;

(j) cryopreserving the infusion bag comprising the harvested TILpopulation from step (f) using a cryopreservation process; and

(k) administering a therapeutically effective dosage of the thirdpopulation of TILs from the infusion bag in step (g) to the subject orpatient.

In an embodiment, the invention provides a method of treating non-smallcell lung carcinoma (NSCLC) by administering a population of tumorinfiltrating lymphocytes (TILs) to a patient in need thereof, whereinthe method comprises:

(a) testing the patient's tumor for PD-L1 expression and tumorproportion score (TPS) of PD-L1,

(b) testing the patient for the absence of one or more driver mutations,wherein the driver mutation is selected from the group consisting of anEGFR mutation, an EGFR insertion, an EGFR exon 20 mutation, a KRASmutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1mutation), a ROS1 fusion, a RET mutation, a RET fusion, an ERBB2mutation, an ERBB2 amplification, a BRCA mutation, a MAP2K1 mutation,PIK3CA, CDKN2A, a PTEN mutation, an UMD mutation, an NRAS mutation, aKRAS mutation, an NF1 mutation, a MET mutation, a MET splice and/oraltered MET signaling, a TP53 mutation, a CREBBP mutation, a KMT2Cmutation, a KMT2D mutation, an ARID1A mutation, a RB1 mutation, an ATMmutation, a SETD2 mutation, a FLT3 mutation, a PTPN11 mutation, a FGFR1mutation, an EP300 mutation, a MYC mutation, an EZH2 mutation, a JAK2mutation, a FBXW7 mutation, a CCND3 mutation, and a GNA11 mutation,

(c) determining that the patient has a TPS score for PD-L1 of less thanabout 1% and determining that the patient also has no driver mutations,

(d) obtaining and/or receiving a first population of TILs from a tumorresected from the subject or patient by processing a tumor sampleobtained from the subject into multiple tumor fragments;

(e) adding the first population of TILs into a closed system;

(f) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 to produce a secondpopulation of TILs, wherein the first expansion is performed in a closedcontainer providing a first gas-permeable surface area, wherein thefirst expansion is performed for about 3-14 days to obtain the secondpopulation of TILs, wherein the second population of TILs is at least50-fold greater in number than the first population of TILs, and whereinthe transition from step (e) to step (f) occurs without opening thesystem;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, OKT-3, andantigen presenting cells (APCs), to produce a third population of TILs,wherein the second expansion is performed for about 7-14 days to obtainthe third population of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (f) to step (g) occurswithout opening the system;

(h) harvesting therapeutic population of TILs obtained from step (d),wherein the transition from step (d) to step (e) occurs without openingthe system; and

(i) transferring the harvested TIL population from step (e) to aninfusion bag, wherein the transfer from step (e) to (f) occurs withoutopening the system;

(j) cryopreserving the infusion bag comprising the harvested TILpopulation from step (f) using a cryopreservation process; and

(k) administering a therapeutically effective dosage of the thirdpopulation of TILs from the infusion bag in step (g) to the subject orpatient.

In an embodiment, the invention provides a method of treating non-smallcell lung carcinoma (NSCLC) by administering a population of tumorinfiltrating lymphocytes (TILs) to a patient in need thereof, whereinthe method comprises:

(a) testing the patient's tumor for PD-L1 expression and tumorproportion score (TPS) of PD-L1,

(b) testing the patient for the absence of one or more driver mutations,wherein the driver mutation is selected from the group consisting of anEGFR mutation, an EGFR insertion, a KRAS mutation, a BRAF mutation, anALK mutation, a c-ROS mutation (ROS1 mutation), a ROS1 fusion, a RETmutation, or a RET fusion,

(c) determining that the patient has a TPS score for PD-L1 of about 1%to about 49% and determining that the patient also has no drivermutations,

(d) obtaining and/or receiving a first population of TILs from a tumorresected from the subject or patient by processing a tumor sampleobtained from the subject into multiple tumor fragments;

(e) adding the first population of TILs into a closed system;

(f) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 to produce a secondpopulation of TILs, wherein the first expansion is performed in a closedcontainer providing a first gas-permeable surface area, wherein thefirst expansion is performed for about 3-14 days to obtain the secondpopulation of TILs, wherein the second population of TILs is at least50-fold greater in number than the first population of TILs, and whereinthe transition from step (e) to step (f) occurs without opening thesystem;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, OKT-3, andantigen presenting cells (APCs), to produce a third population of TILs,wherein the second expansion is performed for about 7-14 days to obtainthe third population of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (f) to step (g) occurswithout opening the system;

(h) harvesting therapeutic population of TILs obtained from step (d),wherein the transition from step (d) to step (e) occurs without openingthe system; and

(i) transferring the harvested TIL population from step (e) to aninfusion bag, wherein the transfer from step (e) to (f) occurs withoutopening the system;

(j) cryopreserving the infusion bag comprising the harvested TILpopulation from step (f) using a cryopreservation process; and

(k) administering a therapeutically effective dosage of the thirdpopulation of TILs from the infusion bag in step (g) to the subject orpatient.

In an embodiment, the invention provides a method of treating non-smallcell lung carcinoma (NSCLC) by administering a population of tumorinfiltrating lymphocytes (TILs) to a patient in need thereof, whereinthe method comprises:

(a) testing the patient's tumor for PD-L1 expression and tumorproportion score (TPS) of PD-L1,

(b) testing the patient for the absence of one or more driver mutations,wherein the driver mutation is selected from the group consisting of anEGFR mutation, an EGFR insertion, a KRAS mutation, a BRAF mutation, anALK mutation, a c-ROS mutation (ROS1 mutation), a ROS1 fusion, a RETmutation, or a RET fusion,

(c) determining that the patient has a TPS score for PD-L1 of less thanabout 1% and determining that the patient also has no driver mutations,

(d) obtaining and/or receiving a first population of TILs from a tumorresected from the subject or patient by processing a tumor sampleobtained from the subject into multiple tumor fragments;

(e) adding the first population of TILs into a closed system;

(f) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 to produce a secondpopulation of TILs, wherein the first expansion is performed in a closedcontainer providing a first gas-permeable surface area, wherein thefirst expansion is performed for about 3-14 days to obtain the secondpopulation of TILs, wherein the second population of TILs is at least50-fold greater in number than the first population of TILs, and whereinthe transition from step (e) to step (f) occurs without opening thesystem;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, OKT-3, andantigen presenting cells (APCs), to produce a third population of TILs,wherein the second expansion is performed for about 7-14 days to obtainthe third population of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (f) to step (g) occurswithout opening the system;

(h) harvesting therapeutic population of TILs obtained from step (d),wherein the transition from step (d) to step (e) occurs without openingthe system; and

(i) transferring the harvested TIL population from step (e) to aninfusion bag, wherein the transfer from step (e) to (f) occurs withoutopening the system;

(j) cryopreserving the infusion bag comprising the harvested TILpopulation from step (f) using a cryopreservation process; and

(k) administering a therapeutically effective dosage of the thirdpopulation of TILs from the infusion bag in step (g) to the subject orpatient.

In another embodiment, the invention provides a method for treating asubject with cancer comprising administering to the subject atherapeutically effective dosage of the therapeutic TIL populationdescribed herein.

In another embodiment, the invention provides a method for treating asubject with cancer comprising administering to the subject atherapeutically effective dosage of the TIL composition describedherein.

In another embodiment, the invention provides the method for treating asubject with cancer described herein modified such that prior toadministering the therapeutically effective dosage of the therapeuticTIL population and the TIL composition described herein, respectively, anon-myeloablative lymphodepletion regimen has been administered to thesubject.

In another embodiment, the invention provides the method for treating asubject with cancer described herein modified such that thenon-myeloablative lymphodepletion regimen comprises the steps ofadministration of cyclophosphamide at a dose of 60 mg/m2/day for twodays followed by administration of fludarabine at a dose of 25 mg/m2/dayfor five days.

In another embodiment, the invention provides the method for treating asubject with cancer described herein modified to further comprise thestep of treating the subject with a high-dose IL-2 regimen starting onthe day after administration of the TIL cells to the subject.

In another embodiment, the invention provides the method for treating asubject with cancer described herein modified such that the high-doseIL-2 regimen comprises 600,000 or 720,000 IU/kg administered as a15-minute bolus intravenous infusion every eight hours until tolerance.

In another embodiment, the invention provides the method for treating asubject with cancer described herein modified such that the cancer is asolid tumor.

In another embodiment, the invention provides the method for treating asubject with cancer described herein modified such that the cancer ismelanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer(NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused byhuman papilloma virus, head and neck cancer (including head and necksquamous cell carcinoma (HNSCC)), glioblastoma (including GBM),gastrointestinal cancer, renal cancer, or renal cell carcinoma.

In another embodiment, the invention provides the method for treating asubject with cancer described herein modified such that the cancer ismelanoma, HNSCC, cervical cancers, NSCLC, glioblastoma (including GBM),and gastrointestinal cancer.

In another embodiment, the invention provides the method for treating asubject with cancer described herein modified such that the cancer ismelanoma.

In another embodiment, the invention provides the method for treating asubject with cancer described herein modified such that the cancer isHNSCC.

In another embodiment, the invention provides the method for treating asubject with cancer described herein modified such that the cancer is acervical cancer.

In another embodiment, the invention provides the method for treating asubject with cancer described herein modified such that the cancer isNSCLC.

In another embodiment, the invention provides the method for treating asubject with cancer described herein modified such that the cancer isglioblastoma (including GBM).

In another embodiment, the invention provides a method for treating asubject with cancer described herein modified such that the cancer isgastrointestinal cancer.

In another embodiment, the invention provides a method for treating asubject with cancer described herein modified such that the cancer is ahypermutated cancer.

In another embodiment, the invention provides a method for treating asubject with cancer described herein modified such that the cancer is apediatric hypermutated cancer.

In another embodiment, the invention provides a therapeutic TILpopulation described herein for use in a method for treating a subjectwith cancer comprising administering to the subject a therapeuticallyeffective dosage of the therapeutic TIL population.

In another embodiment, the invention provides a TIL compositiondescribed herein for use in a method for treating a subject with cancercomprising administering to the subject a therapeutically effectivedosage of the TIL composition.

In another embodiment, the invention provides a therapeutic TILpopulation described herein or the TIL composition described hereinmodified such that prior to administering to the subject thetherapeutically effective dosage of the therapeutic TIL populationdescribed herein or the TIL composition described herein, anon-myeloablative lymphodepletion regimen has been administered to thesubject.

In another embodiment, the invention provides a therapeutic TILpopulation or the TIL composition described herein modified such thatthe non-myeloablative lymphodepletion regimen comprises the steps ofadministration of cyclophosphamide at a dose of 60 mg/m2/day for twodays followed by administration of fludarabine at a dose of 25 mg/m2/dayfor five days.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified to furthercomprise the step of treating patient with a high-dose IL-2 regimenstarting on the day after administration of the TIL cells to thepatient.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified such that thehigh-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg administeredas a 15-minute bolus intravenous infusion every eight hours untiltolerance.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified such that thecancer is a solid tumor.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified such that thecancer is melanoma, ovarian cancer, cervical cancer, non-small-cell lungcancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancercaused by human papilloma virus, head and neck cancer (including headand neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM),gastrointestinal cancer, renal cancer, or renal cell carcinoma.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified such that thecancer is melanoma, HNSCC, cervical cancers, NSCLC, glioblastoma(including GBM), and gastrointestinal cancer.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified such that thecancer is melanoma.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified such that thecancer is HNSCC.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified such that thecancer is cervical cancer.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified such that thecancer is NSCLC.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified such that thecancer is glioblastoma.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified such that thecancer is gastrointestinal cancer.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified such that thecancer is a hypermutated cancer.

In another embodiment, the invention provides a therapeutic TILpopulation or a TIL composition described herein modified such that thecancer is a pediatric hypermutated cancer.

In another embodiment, the invention provides the use of a therapeuticTIL population described herein in a method of treating cancer in asubject comprising administering to the subject a therapeuticallyeffective dosage of the therapeutic TIL population.

In another embodiment, the invention provides the use of a TILcomposition described in any of the preceding paragraphs in a method oftreating cancer in a subject comprising administering to the subject atherapeutically effective dosage of the TIL composition.

In another embodiment, the invention provides the use of a therapeuticTIL population described herein or a TIL composition described herein ina method of treating cancer in a patient comprising administering to thepatient a non-myeloablative lymphodepletion regimen and thenadministering to the subject the therapeutically effective dosage of thetherapeutic TIL population described in any of the preceding paragraphsor the therapeutically effective dosage of the TIL composition describedherein.

1. Methods of Treating Cancers Based on Driver Mutations

As used herein, the phrases “driver mutation” and/or “actionablemutation” and/or “oncogenic driver mutation” refer to mutations that aretypically considered oncogenic drivers (i.e., cancer drivers or cancerinducers). The presence of one or more of these mutations hastraditionally been the utilized as the target for a targeted therapy.Often, driver mutations are examined and/or analyzed for treatment withtargeted therapeutic moieties, including for example tyrosine kinaseinhibitors (TKIs). Such driver mutations can, in some embodiments,impact or affect response to a first line therapeutic treatment. TILtherapy methods and compositions described herein are effective fortreatment whether such driver mutations are present or absent in thepatient or subject. Such driver mutations can be tested and determinedby any method known in the art, including whole exome sequencing ormethods targeted to the detection of a specific driver mutation.

In some embodiments, the cancer is a cancer that exhibits the presenceor absence of one or more driver mutations. In some embodiments, thecancer exhibits the presence of one or more driver mutations. In someembodiments, a cancer exhibits the absence of one or more drivermutations. In some embodiments, a cancer has been analyzed for theabsence or presence of one or more driver mutations. In someembodiments, the one or more driver mutations are not present. In someembodiments, a cancer treatment is independent of the presence orabsence of one or more driver mutations. In some embodiments, the cancerexhibits one or more driver mutations selected from the group consistingof an EGFR mutation, an EGFR insertion, EGFR exon20, a KRAS mutation, aBRAF-mutation, a BRAF V600E mutation, a BRAF V600K mutation, a BRAF V600mutation, an ALK mutation, a c-ROS mutation (ROS1-mutation), a ROS1fusion, a RET mutation, a RET fusion, an ERBB2 mutation, an ERBB2amplification, a BRCA mutation, a MAP2K1 mutation, PIK3CA, CDKN2A, aPTEN mutation, an UMD mutation, an NRAS mutation, a KRAS mutation, anNF1 mutation, a MET mutation, a MET splice and/or altered MET signaling,a TP53 mutation, a CREBBP mutation, a KMT2C mutation, a KMT2D mutation,an ARID1A mutation, a RB1 mutation, an ATM mutation, a SETD2 mutation, aFLT3 mutation, a PTPN11 mutation, a FGFR1 mutation, an EP300 mutation, aMYC mutation, an EZH2 mutation, a JAK2 mutation, a FBXW7 mutation, aCCND3 mutation, and a GNA11 mutation. In some embodiments, the cancerexhibits a PD-L1 TPS of <1% and has a predetermined absence of one ormore driver mutations.

In some embodiments, a cancer is an cancer that is not indicated fortreatment by an EGFR inhibitor, a BRAF inhibitor, an ALK inhibitor, ac-Ros inhibitor, a RET inhibitor, an ERBB2 inhibitor, BRCA inhibitor, aMAP2K1 inhibitor, PIK3CA inhibitor, CDKN2A inhibitor, a PTEN inhibitor,an UMD inhibitor, an NRAS inhibitor, a KRAS inhibitor, an NF1 inhibitor,MET inhibitor a TP53 inhibitor, a CREBBP inhibitor, a KMT2C inhibitor, aKMT2D mutation, an ARID1A mutation, a RB1 inhibitor, an ATM inhibitor, aSETD2 inhibitor, a FLT3 inhibitor, a PTPN11 inhibitor, a FGFR1inhibitor, an EP300 inhibitor, a MYC inhibitor, an EZH2 inhibitor, aJAK2 inhibitor, a FBXW7 inhibitor, a CCND3 inhibitor, and a GNA11inhibitor.

In some embodiments, a cancer exhibits a PD-L1 TPS of <1% and is notindicated for treatment by an EGFR inhibitor, a BRAF inhibitor, an ALKinhibitor, a c-Ros inhibitor, a RET inhibitor, an ERBB2 inhibitor, BRCAinhibitor, a MAP2K1 inhibitor, PIK3CA inhibitor, CDKN2A inhibitor, aPTEN inhibitor, an UMD inhibitor, an NRAS inhibitor, a KRAS inhibitor,an NF1 inhibitor, MET inhibitor a TP53 inhibitor, a CREBBP inhibitor, aKMT2C inhibitor, a KMT2D mutation, an ARID1A mutation, a RB1 inhibitor,an ATM inhibitor, a SETD2 inhibitor, a FLT3 inhibitor, a PTPN11inhibitor, a FGFR1 inhibitor, an EP300 inhibitor, a MYC inhibitor, anEZH2 inhibitor, a JAK2 inhibitor, a FBXW7 inhibitor, a CCND3 inhibitor,and a GNA11 inhibitor

In some embodiments, the cancer is NSCLC, and an EGFR mutation resultsin tumor transformation from NSCLC to small cell lung cancer (SCLC).

In some embodiments, a cancer (or a biopsy thereof) exhibits high-tumormutational burden (high-TMB; >10 mut/kb) and/or microsatelliteinstability high (MSI-high). In some embodiments, the cancer (or abiopsy thereof) exhibits high-tumor mutational burden (high-TMB; >10mut/kb). In some embodiments, a cancer (or a biopsy thereof) exhibitsmicrosatellite instability high (MSI-high). Methods and systems forevaluating tumor mutational burden are known in the art. Exemplarydisclosures of such methods and systems can be found in U.S. Pat. No.9,792,403, U.S. Patent Application Publication No. US 2018/0363066 A1,International Patent Application Publication Nos. WO 2013/070634 A1 andWO 2018/106884 A1, and Metzker, Nature Biotechnol. Rev. 2010, 11, 31-46,each of which is incorporated by reference herein.

In some embodiments, an EGFR mutation includes, for example, but is notlimited to T790M, Ex19Del, L858R, Exon 20 insertion, delE709-T710insD,I744_K745insKIPVAI, K745_E746insTPVAIK, E709X, E709K, E709A, Exon 18deletion, G719X, G719A, G719S, L861Q, S768I, L747P, A763_764insFQEA,D770_N771insNPG, A763_764insFQEA, P772_H773insDNP exon 20 insertion,H773_V774insNPH exon 20 insertion, S768I, D770_N771insSVD,V769_D770InsASV, p.K745_E746insIPVAIK, p.K745_E746insTPVAIK,p.I744_K745insKIPVAI, D770_N771insNPG, P772_H773insPNP,A763_Y764insFQEA, and/or EGFR kinase domain duplication (EGFR-KDD). Insome embodiments, an EGFR mutation is selected from the group consistingof T790M, Ex19Del, L858R, Exon 20 insertion, delE709-T710insD,I744_K745insKIPVAI, K745 E746insTPVAIK, E709X, E709K, E709A, Exon 18deletion, G719X, G719A, G719S, L861Q, S768I, L747P, A763_764insFQEA,D770_N771insNPG, A763_764insFQEA, P772_H773insDNP exon 20 insertion,H773_V774insNPH exon 20 insertion, S768I, D770_N771insSVD,V769_D770InsASV, p.K745_E746insIPVAIK, p.K745 E746insTPVAIK,p.I744_K745insKIPVAI, D770_N771insNPG, P772_H773insPNP,A763_Y764insFQEA, and EGFR kinase domain duplication (EGFR-KDD).

In some embodiments, an EGFR mutation is a double mutation, including,but not limited to, L858R/T790M, Ex19Del/T790M, G719X/L861Q, G719X/S768I(or S768I/G719X), S768I/L858R, L858R/E709A, and/or E746T751delinsA+T790M. In some embodiments, an EGFR mutation is a doublemutation selected from group consisting of L858R/T790M, Ex19Del/T790M,G719X/L861Q, G719X/S768I (or S768I/G719X), S768I/L858R, L858R/E709A, andE746_T751delinsA+T790M. Additional properties and methods regarding EGFRmutations are provided in International Patent Application PublicationNo. WO 2010/020618 A1, which is incorporated by referenced herein.

In some embodiments, the ALK mutation includes, but not limited to,EML4-ALK Variant 1 (AB274722.1; BAF73611.1), EML4-ALK Variant 2(AB275889.1; BAF73612.1), EML4-ALK Variant 3a (AB374361.1; BAG55003.1),EML4-ALK Variant 3b (AB374362.1; BAG55004.1), EML4-ALK Variant 4(AB374363.1; BAG75147.1), EML4-ALK Variant 5a (AB374364.1; BAG75148.1),EML4-ALK Variant 5b (AB374365.1; BAG75149.1), EML4-ALK Variant 6(AB462411.1; BAH57335.1), EML4-ALK Variant 7 (AB462412.1; BAH57336.1),K1F5B-ALK (AB462413.1; BAH57337.1), NPM-ALK, TPM3-ALK, TFGXL-ALK,TEGL-ALK, TFGS-ALK, A11C-ALK, CLTC-ALK, MSN-ALK, TPM4-ALK, MYH9-ALK,RANBP2-ALK, AL017-ALK, and CARS-ALK (see, for example. Pulford et al.,(2004) J. Cell. Physiol. 199:330-358). In addition, a skilled artisanwill understand that ALK kinase variants can arise depending upon theparticular fusion event between an ALK kinase and its fusion partner(e.g., EML4 can fuse at least exon 2, 6a, 6b, 13, 14, and/or 15, asdescribed, for example, in Horn and Pao, J. Clin. Oncol. 2009, 27,4247-4253, the disclosure of which is incorporated by reference herein.

Additional examples of ALK mutations are described in U.S. Pat. Nos.9,018,230 and 9,458,508, the disclosures of which are incorporated byreference herein.

In some embodiments, the ROS1 mutation of the present invention is aROS1 fusion, where a portion of the ROS1 polypeptide that includes thekinase domain of the ROS1 protein (or polynucleotide encoding the same)fused to all or a portion of another polypeptide (or polynucleotideencoding the same) and where the name of that second polypeptide orpolynucleotide is named in the fusion. In some embodiments, the ROS1mutation is determined as ROS1-fusion protein (e.g., by IHC) and/orROS-fusion gene (e.g. by FISH), and/or ROS1 mRNA (e.g. by qRT-PCR),preferably indicative of a ROS1-fusion protein selected from the groupconsisting of SLC34A2-ROS1 (SLC34A2 exons 13del2046 and 4 fused to ROS1exons 32 and 34), CD74-ROS1 (CD74 exon 6 fused to ROS1 exons 32 and 34),EZR-ROS1 (EZR exon 10 fused to ROS1 exon 34), TPM3-ROS1 (TPM3 exon 8fused to ROS1 exon 35), LRIG3-ROS1 (LRIG3 exon 16 fused to ROS1 exon35), SDC4-ROS1 (SDC4 exon 2 and 4 fused to ROS1 exon 32 and SDC4 exon 4fused to ROS1 exon 34), GOPC-ROS1, also known as FIG-ROS1, (GOPC exon 8fused to ROS1 exon 35 and GOPC exon 4 fused to ROS1 exon 36), andG2032R, also known as ROS1G2032R.

Additional disclosures of ROS1 mutations and a ROS fusion have beenprovided in U.S. Patent Application Publication Nos. US 2010/0221737 A1,US 2015/0056193 A1, and US 2010/0143918 A1, and in International PatentApplication Publication No WO 2010/093928 A1, each of which areincorporated by reference herein. In some embodiments, the RET mutationis a RET fusion or point mutation.

In some embodiments, a RET point mutation includes but is not limited toH6650, K666E, K666M, S686N, G691S, R694Q, M700L, V706M, V706A, E713K,G736R, G748C, A750P, S765P, P766S, E768Q, E768D, L769L, R770Q, D771N,N777S, V7781, Q781R, L790F, Y791F, Y791N, V804L, V804M, V804E, E805K,E806C, Y806E, Y806F, Y8065, Y806G, Y806C, E818K, S819I, G823E, Y826M,R833C, P841L, P841P, E843D, R844W, R844Q, R844L, M848T, 1852M A866W,R873W, A876V, L881V, A883F, A883S, A883T, E884K, R886W, S891A, R8970,D898V, E901K, 5904F, 5904C2, K907E, K907M, R908K, G911D, R912P, R912QM918T, M918V, M918L6, A919V, E921K, S922P, S922Y, T930M, F961L, R972G,R982C, M1009V, D1017N, V10416, and M1064T.

In some embodiments, a RET fusion is a fusion between RET and a fusionpartner that is selected from the group consisting of BCR, BCR, CLIP 1,KIFSB, CCDCl6, PTClex9, NCOA4, TRIM33, ERC1, FGFRIOP, MBD1, RAB61P2,PRKARIA, TRIM24, KTN1, GOLGA5, HOOK3, KIAA1468, TRIM27, AKAP13, FKBP15,SPECCIL, TBL1XR1, CEP55, CUX1, ACBD5, MYH13, PIBF1, KIAA1217, and MPRIP.

Additional disclosures of a RET mutations has been provided in U.S.patent Ser. No. 10/035,789, which is hereby incorporated by reference intheir entirety.

In some embodiments, a BRAF mutation is BRAF V600E/K mutation. In otherembodiments, the BRAF mutation is a non-V600E/K mutation.

In some embodiments, a non-V600E/K BRAF mutation is a kinase-activatedmutation, a kinase-impaired mutation, or a kinase-unknown mutation, andcombinations thereof. In some embodiments, a kinase-activated mutationis selected from the group consisting of R4621, 1463S, G464E, G464R,G464V, G466A, G469A, N58 is, E586K, F595L, L597Q, L597R, L5975, L597V,A598V, T599E, V600R, K601E, 5602D, A728V, and combinations thereof. Insome embodiments, a kinase-impaired mutation is selected from the groupconsisting of G466E, G466R, G466V, Y472C, K483M, D594A, D594E, D594G,D594H, D594N, D594V, G596R, T599A, 5602A, and combinations thereof. Insome embodiments, a kinase-unknown mutation is selected from the groupconsisting of T4401, 5467L, G469E, G469R, G4695, G469V, L584F, L588F,V600 K6OldelinsE, 56051, Q609L, E611Q, and combinations thereof. In someembodiments, the non-V600E/K BRAF mutation is selected from the groupconsisting of D594, G469, K601E, L597, T599 duplication, L485W, F247L,G466V, BRAF fusion, BRAF-AGAP3 rearrangement, BRAF exon 15 slicevariant, and combinations thereof.

In some embodiments, a Met mutation includes point mutation, deletionmutation, insertion mutation, inversion, aberrant splicing, missensemutation, or gene magnification that causes the increase of at least onebioactivity of c-Met protein, the tyrosine kinase activity such asimproved, receptor homolog dimerization Ligand binding of formation,enhancing of body and heterodimer etc. The Met mutation can be locatedat any part of c-Met genes. In one embodiment, the mutation is in thekinase domain of c-Met protein encoded by the c-MET gene. In someembodiments, the c-Met mutations are point mutation at N375, V13, V923,R175, V136, L229, 5323, R988, 51058/T1010 and E168.

In some embodiments, an ERBB2 mutation is a point mutation in the aminoacid sequence of ERBB2. In some embodiments, the point mutation of ERBB2is one that causes amino acid substitutions, causes mRNA splicing, or isa point mutation in the upstream region. Wherein the mutation comprisesa nucleotide mutation causing at least one amino acid substitutionselected from the group consisting of Q568E, P601R, I628M, P885S, R143Q,R434Q, and E874K.

In some embodiments, an ERBB2 mutation is ERBB2 amplification. In someembodiments, the ERBB2 amplification includes point mutation selectedfrom the group consisting of V659E, G309A, G309E, S310F, D769H, D769Y,V777L, P780ins, P780-Y781insGSP, V842I, R896C, K753E, and L755S and canbe detected by polymerase chain reaction or other sequencing techniquesknown in the art, such as those described in Bose, et al., CancerDiscov. 2013, 3(2), 224-237; and Zuo, et al. Clin Cancer Res. 2016,22(19), 4859-4869, the disclosures of which are incorporated byreference herein.

In some embodiments, a BRCA mutation is a mutation in BRCA1 and/orBRCA2, preferably BRCA1, and/or in one or more other genes of which theprotein product associates with BRCA1 and/or BRCA2 at DNA damage sites,including ATM, ATR, Chk2, H2AX, 53BP1, NFBD1, Mre11, Rad50, Nibrin,BRCA1-associated RING domain (BARD1), Abraxas, and MSH2. A mutation inone or more of these genes may result in a gene expression pattern thatmimics a mutation in BRCA1 and/or BRCA2.

In certain embodiments, a BRCA mutation comprises a non-synonymousmutation. In some embodiments, a BRCA mutation comprises a nonsensemutation. In some embodiments, the BRCA mutation comprises a frameshiftmutation. In some embodiments, the BRCA mutation comprises a splicingmutation. In some embodiments, a BRCA mutation is expressed as a mutantmRNA and ultimately a mutant protein. In some embodiments, a BRCA1/2protein is functional. In other embodiments, a BRCA1/2 protein hasreduced activity. In other embodiments, a BRCA1/2 protein isnon-functional.

As used herein with regard to substitutions, the “=” sign with regard tomutations generally refers to synonymous substitutions, silent codons,and/or silent substitutions. In particular, a synonymous substitution(also called a silent substitution or silent codon) refers to thesubstitution of one nucleotide base for another in an exon of a geneencoding a protein, wherein the produced amino acid sequence is notmodified. This is due to the fact that the genetic code is “degenerate”,i.e., that some amino acids are coded for by more than onethree-base-pair codon. Because some of the codons for a given amino acidvary by just one base pair from others coding for the same amino acid, apoint mutation that replaces the wild-type base by one of thealternatives will result in incorporation of the same amino acid intothe elongating polypeptide chain during translation of the gene. In someembodiments, synonymous substitutions and mutations affecting noncodingDNA are often considered silent mutations; however, it is not always thecase that the mutation is silent and without any impact. For example, asynonymous mutation can affect transcription, splicing, mRNA transport,and translation, any of which could alter the resulting phenotype,rendering the synonymous mutation non-silent. The substrate specificityof the tRNA to the rare codon can affect the timing of translation, andin turn the co-translational folding of the protein. This is manifestedin the codon usage bias that has been observed in many species. Anonsynonymous substitution/mutation results in a change in amino acidthat may be arbitrarily classified as conservative (a change to an aminoacid with similar physiochemical properties), semi-conservative (e.g.negatively to positively charged amino acid), or radical (vastlydifferent amino acid). In some embodiments, the BRCA mutation is a BRCA1mutation that includes, but is not limited to P871L, K1183R, D693N,S1634G, E1038G, S1040N, S694=(=: silence codon), M1673I, Q356R, S1436=,L771=, K654Sfs*47, S198N, R496H, R841W, R1347G, H619N, S1533I, L30=,A622V, Y655Vfs*18, R496C, E597K, R1443*, E23Vfs*17, L30F, E111Gfs*3,K339Rfs*2, L512F, D693N, P871S, 51140G, Q1240*, P1770S, R7=, L52F,T176M, A224S, L347=, S561F, E597*, K820E, K893Rfs*107, E962K, M1014I,R1028H, E1258D, E1346K, R1347T, L1439F, H1472R, Q1488*, 51572C, E1602K,R1610C, L1621=, Q1625*, Q1625=, D1754N, R1772Q, R1856*, and anycombination thereof.

In some embodiments, a BRCA mutation is a BRCA2 mutation that includes,but is not limited to V2466A, N289H, N991D, S455=(=: silence codon),N372H, H743=, V1269=, S2414=, V2171=, L1521=, T3033Nfs*11, K1132=,T3033Lfs*29, R2842C, N1784Tfs*7, K3326*, K3326*, D1420Y, I605Yfs*9,I3412V, A2951T, T3085Nfs*26, R2645Nfs*3, S1013*, T1915M, F3090=, V3244I,A1393V, R2034C, L1356=, E2981Rfs*37, N1784Kfs*3, K3416Nfs*11,K1691Nfs*15, S1982Rfs*22, and any combination thereof.

In some embodiments, an NRAS mutation includes but is not limited toE63K, Q61R, Q61K, G12D, G13D, Q61R, Q61L, Q61K, G12S, G12C, G13R, Q61H,G12V, G12A, Q61L, G13V, Q61H, Q61H, G12R, G13C, Q61P, G13S, G12D, G13A,G13D, A18T, Q61X, G60E, G12S, Q61=(=: silence codon), Q61E, Q61R, A146T,A59T, A59D, Q61=, R68T, A146T, G12A, E62Q, G75=, A91V, and anycombination thereof.

E132KIn some embodiments, a PIK3CA mutation includes substitutionmutations, deletion mutations, and insertion mutations. In someembodiments, mutations occur in PIK3CA's helical domain and in itskinase. In other embodiments, in PIK3CA's P85BD domain. In someembodiments, the PIK3CA mutation is in exon 1, 2, 4, 5, 7, 9, 13, 18,and 20. In some embodiments, the PIK3CA mutation is in exons 9 and 20.In yet other embodiments, the PIK3CA mutation is a combination of theany mutations listed above. Any combination of these exons can betested, optionally in conjunction with testing other exons. Testing formutations can be done along the whole coding sequence or can be focusedin the areas where mutations have been found to cluster. Particularhotspots of mutations occur at nucleotide positions 1624, 1633, 1636,and 3140 of a PIK3CA coding sequence.

In some embodiments, the size of a PIK3CA mutation is small, rangingfrom 1 to 3 nucleotides. In some embodiments, the PIK3CA mutationsinclude, but are not limited to G1624A, G1633A, C1636A, A3140G, G113A,T1258C, G3129T, C3139T, E542K, E545K, Q546R, H1047L, H1047R and G2702T.

In some embodiments, a MAP2K1 mutation is a somatic MAP2K1 mutation,optionally a MAP2K1 mutation that upregulates MEK1 levels. In someembodiments, the MAP2K1 mutation is a mutation in one or more genesassociated with the RAS/MAPK pathway, comprising: HRAS, KRAS, NRAS,ARAF, BRAF, RAF1, MAP2K2, MAPK1, MAPK3, MAP3K3. In certain embodiments,the MAP2K1 mutation is in one or more genes selected from the groupconsisting of RASA, PTEN, ENG, ACVRL1, SMAD4, GDF2 or combinationsthereof.

In some embodiments, a MAP2K1 mutation includes, but is not limited to,P124S, Q56P, K57N, E203K, G237*, P124L, G128D, D67N, K57E, E102 I103del,C121S, K57T, K57N, Q56P, P124L, K57N, G128V, Q58 E62del, F53L, I126=,I103_K104del, and any combination thereof.

In some embodiments, a KRAS mutation comprises a non-synonymousmutation. In some embodiments, a KRAS mutation comprises a nonsensemutation. In some embodiments, a KRAS mutation comprises a frameshiftmutation. In some embodiments, a KRAS mutation comprises a splicingmutation. In some embodiments, a KRAS mutation is expressed as a mutantmRNA and ultimately a mutant protein. In some embodiments, a mutatedKRAS protein is functional. In other embodiments, a mutated KRAS proteinhas reduced activity. In other embodiments, a mutated KRAS protein isnon-functional.

In some embodiments, a KRAS mutation includes but is not limited toG12D, G12V, G13D, G12C, G12A, G12S, G12R, G13C, Q61H, A146T, Q61R, Q61H,Q61L, G13S, A146V, Q61K, G13R, G12F, K117N, G13A, G13V, A59T, V14I,K117N, Q22K, Q61P, A146P, G13D, L19F, L19F, Q61K, G12V, G60=, G12=,G13=, A18D, T58I, Q61E, E63K, G12L, G13V, A59G, G60D, G10R, G10dup,D57N, A59E, V14G, D33E, G12I, G13dup, and any combination thereof,wherein=is indicative of silence coding.

In some embodiments, a NF1 mutation includes substitution mutations,deletion mutations, missense mutations, aberrant splicing mutations, andinsertion mutations. In some embodiments, the NF1 mutation is a loss offunction (LOF) mutation. In some embodiments, the NF1 mutation isselected from the group consisting of R1947X (C5839T), R304X, exon 37mutation, exon 4b mutation, exon 7 mutation, exon 10b mutation, and exon10c mutation (e.g., 1570G4T, E524X).

In some embodiments, a CDKN2A mutation includes but is not limited toR24P, D108G, D108N, D108Y, G125R, P114L, R80*, R58*, H83Y, W110*, P114L,E88*, W110*, E120*, D108Y, D84Y, D84N, E69*, P81L, Q50*, L78Hfs*41,D108N, S12*, P48L, E61*, Y44*, E88K, R80*, D84G, L16Pfs*9, Y129*, D108H,A148T, A36G, A102V, W15*, H83R, A57V, E33*, D74Y, A76V, E153K, D74N,H83D, V82M, R58*, Y129*, E119*, Y44*, D74A, T18_A19dup, Y44Lfs*76,L32_L37del, V28_E33del, D14_L16del, A68T, or any combination thereof.

In some embodiments, a PTEN mutation comprises a non-synonymousmutation. In some embodiments, the PTEN mutation comprises a nonsensemutation. In some embodiments, the PTEN mutation comprises a frameshiftmutation. In some embodiments, the PTEN mutation comprises a splicingmutation. In some embodiments, the mutated PTEN is expressed as an mRNAand ultimately a protein. In some embodiments, the mutated PTEN proteinis functional. In other embodiments, the mutated PTEN protein hasreduced activity. In other embodiments, the mutated PTEN protein isnon-functional. In some embodiments, the PTEN mutation includes, but isnot limited to, R130Q, R130G, T319*, R233*, R130*, K267Rfs*9,N323Mfs*21, N323Kfs*2, R173C, R173H, R335*, Q171*, Q245*, E7*,D268Gfs*30, R130Q, Q214*, R130L, C136R, Q298*, Q17*, H93R, P248Tfs*5,I33del, R233*, E299*, G132D, Y68H, T319Kfs*24, N329Kfs*14, V166Sfs*14,V290*, T319Nfs*6, R142W, P38S, A126T, H61R, F278L, S229*, R130P, G129R,R130Qfs*4, P246L, R130*, G165R, C136Y, R173C, I101T, Y155C, D92E,K164Rfs*3, N184Efs*6, G129E, R130G, G36R, F341V, H123Y, C124S,M35VG127E, G165E and any combination thereof.

In some embodiments, a TP53 mutation includes, but is not limited to,R175H, G245S, R248Q, R248W, R249S, R273C, R273H, R282W, C135Y, C141Y,P151S, V157F, R158L, Y163C, V173L, V173M, C176F, H179R, H179Y, H179Q,Y205C, Y220C, Y234C, M237I, C238Y, S241F, G245D, G245C, R248L, R249M,V272M, R273L, P278L, R280T, E285K, E286K, R158H, C176Y, I195T, G214R,G245V, G266R, G266E, P278S, R280K, or any combination thereof. In somefurther embodiments, the TP53 mutation is selected from the groupconsisting of: G245S; R249S; R273C; R273H; C141Y, V157F, R158L, Y163C,V173L, V173M, Y205C, Y220C, G245C, R249M, V272M, R273L, and E286K. Insome embodiments, the TP53 mutation includes one or more of themutations above.

In some embodiments, a CREBBP mutation includes, but is not limited to,R1446C, R1446H, S1680del, I1084Sfs*15, P1948L, I1084Nfs*3, ?R386*,S893L, R1341*, P1423Lfs*36, P1488L, Y1503H, R1664C, A1824T, R1173*,R1360*, Y1450C, H2228D, S71L, P928=, D1435N, W1502C, Y1503D, R483*,R601Q, S945L, R1103*, R1288W, R1392*, C1408Y, D1435G, R1446L, H1485Y,Q1491K, Q96*, L361M, L524Wfs*6, Q540*, Q1073*, A1100V, R1169C, C1237Y,R1347W, G1411E, W1472C, I1483F, P1488T, R1498*, Y1503F, Q1856*, R1985C,R2104C, S2328L, V2349=, S2377L, and any combination thereof.

In some embodiments, a KMT2C mutation includes, but is not limited to,D348N, P350=, R380L, C391*, P309S, C988F, Y987H, S990G, K2797Rfs*26,V346=, R894Q, R284Q, S806=, R1690=, P986=, A1685S, G315S, Q755*, R909K,T316S, S772L, G838S, L291F, P335=, C988F, Q2680=, E765G, K339N, Y816*,R526P, N729D, G845E, I817Nfs*11, G892R, C1103*, S3660L, F4496Lfs*21,G315C, R886C, D348N, S793=, V919L, R2481S, R2884*, R4549C, M305Dfs*28,T316S, P377=, I455M, T8201, S965=, S730Y, P860S, Q873Hfs*40, R904*,R2610Q, R4478*, and any combination thereof.

In some embodiments, a KMT2D mutation includes, but is not limited to,L1419P, E640D, E541D, E455D, T2131P, K1420R. P2354Lfs*30, G2493=,Q3612=, I942=, T1195Hfs*17, P4170=, P1194H, G1235Vfs*95, P4563=,P647Hfs*283, L449_P457del, P3557=, Q3603=, R1702*, P648Tfs*2, R5501*,R4198*, R4484*, R83Q, R1903*, R2685*, R4282*, L5326=, R5432W, R2734*,Q2800*, R2830*, Q3745dup, S4010P, R4904*, G5182Afs*61, R5214H, R1615*,Q2380*, R2687*, R2771*, V3089Wfs*30, Q3799Gfs*212, R4536*, R5030C,R5048C, R5432Q, A221Lfs*40, A476T, A2119Lfs*25, P2557L, R2801*, Q3913*,R4420W, G4641=, R5097*, and any combination thereof.

In some embodiments, a ARID1A mutation includes, but is not limited to,For example, subject has a mutation of ARID1A selected from the groupconsisting of a C884* (*: nonsense mutation), E966K, Q1411*, F1720fs(fs: frameshift), G1847fs, C1874fs, D1957E, Q1430, R1721fs, G1255E,G284fs, R1722*, M274fs, G1847fs, P559fs, R1276*, Q2176fs, H203fs,A591fs, Q1322*, S2264*, Q586*, Q548fs, and N756fs.

In some embodiments, a RB1 mutation includes, but is not limited to,R320X, R467X, R579X, R455X, R358X, R251X, R787X, R552X, R255X, R556X,Y790X, Q575X, E323X, R661W, R579*, R455*, R556*. R787*, R661W, R445*,R467*, Q217*, Q471*, W195*, Q395*, 1680T, E137*, R255*, Q344*, Q62*,E440K, A488V, P777Lfs*33, E322K, R656W, G617Rfs*36, C221*, E440*, Q93*,Q504*, E125*, S834*, E323*, Q685*, S829*, W516*, G435*, Q257*, E79*,S567L, V654M, V654Sfs*14, G100Efs*11, K715*, and any combinationthereof.

In some embodiments, an ATM mutation is a mutation in the ATM genesequence including, but is not limited to, 10744A>G; 10744A>G; 11482G>A;IVS3-558A>T; 146C>G; 381delA; IVS8-3delGT; 1028delAAAA; 1120C>T;1930ins16; IVS16+2T>C; 2572T>C; IVS21+1G>A; 3085delA; 3381delTGAC;3602delTT; 4052delT; 4396C>T; 5188C>T; 5290delC; 5546delT; 5791G>CCT;6047A>G; IVS44-1G>T; 6672delGC/6677delTACG; 6736dell 1/6749de17;7159insAGCC; 7671delGTTT; 7705del14; 7865C>T; 7979delTGT; 8177C>T;8545C>T; 8565T>A; IVS64+1G>T; and 9010del28.

In some embodiments of the present invention, a SETD2 mutation is analteration in the gene sequence encoding the SETD2 protein, when thetranscription initiation codon position of the mRNA sequence of NCBIaccession number NM 014159 is set to 1. In some embodiments, the 7558thG (guanine) is substituted with T (thymine), the 4774th C (cytosine) issubstituted by T, the 1210th A (adenine) is substituted by T, the 4883thT is substituted by G, the 5290th C is replaced by T, the 7072th C isreplaced by T, the 4144th G is substituted by T, the 1297 C is replacedby T, the 755th T is replaced by G, the 7261 T is substituted by G, 6700is replaced by T, the 2536th C is substituted by T, the 7438th C isreplaced by T substitution, or there is an insertion of A at position3866, insertion of T at position 6712, insertion of T at position 7572,deletion of the 913th A, deletion of the 5619th C, deletion of bases4603-4604, deletion of the 1st base, deletion of the 1936th C, deletionof the 3094-3118 base, insertion of A in the 5289th position, anddeletion of the 6323-6333 base.

In some embodiments, a FLT3 mutation includes, but is not limited to,(Q569_E648)ins, D835X, (Q569_E648)delins, (D835_I836), D835Y, D835V,D835Y, D835H, T227M, I836del, N676K, D835E, Y597 E598insDYVDFREY, D835E,D835del, F594_D600dup, A680V, D839G, D96=, D835H, V491L, D835E, Q989*,D835V, L561=, I836del, P986Afs*27, D7G, D324N, S451F, D835N, L576P,Y597_E598insDVDFREY, V491L, N841T, D324N, Y572C, R595_L601dup, K663R,N676K, F691L, D835A, I836H, N841K, S993L, L832F, I836M, A66V, and anycombination thereof.

In some embodiments, a PTPN11 mutation includes, but is not limited to,c E76K, A72V, A72T, D61Y, D61V, G60V, E69K, E76G, G507V, S506L, G507A,T73I, E76A, E76Q, S506P, D61N, F71L, E76V, F71L, A72D, V432M, T472M,P495L, N58Y, F285S, S506A, S189A, A465T, R502W, G507R, T511K, D61H,D61G, G507E, G60R, G60A, Q514L, E139D, Y197*, N308D, Q514H, Q514H, N58S,E123D, L206=, A465G, P495S, G507R, and any combination thereof.

In some embodiments, a FGFR1 mutation includes, but is not limited to,N577K, K687E, N577K, D166del, T371M, R476W, T350=, E498K, N577D, D683G,R87C, A154D, N303=, A374V, D550=, S633=, V695L, G728=, R765W, P803S,W19C, P56=, R113C, V149I, S158L, D166dupR220C, N224Kfs*8, D249N, R281W,R281Q, A299S, S424L, S461F, S467F, R506Q, and any combination thereof.

In some embodiments, a EP300 mutation includes, but is not limited to,D1399N, Y1414C, M1470Cfs*26, Y1111*, H2324Pfs*55, R1627W,N2209_Q2213delinsK, Q2268del, L415P, M1470Nfs*3, E1514K, C1201Y, P1452L,S952*, C1164Y, D1399Y, S507G, Q824*, D1507N, H2324Tfs*29, P925T, P1440L,W1466C, P1502L, A1629V, R1645*, N1700Tfs*9, P1869L, Q65*, A171V, R202*,R580Q, A627V, Q1082*, N1236Kfs*2, N1286S, R1312*, R1356*, C1385F,H1451L, R1462*, Y1467N, Y1467H, R1478H, R1627Q, R86*, R370H, R397*,R754C, P842S, I997V, E1014*, and any combination thereof.

In some embodiments, a MYC mutation includes, but is limited to, E61T,E68I, R74Q, R75N, W135E, W136E, V394D, L420P, W96E, V325D, L351P, a MYCprotein with 41 amino acid deleted at the N-terminus (dN2MYC), N26S,S161L, P74L, V7M, F153S, E54D, P246, L164V, P74S, A59V, T73I, P72T,T73A, H374R, P17S, T73N, S264N, P72S, Q52del, S21T, P74A, S107N, P75S,S77P, P261S, P74Q, S190R, A59T, F153C, P75H, T73I, S77F, N11S, S21N,P78L, P72L, N9K, S190N, S267F, T73P, P78S, G105D, S187C, L71M, Q10H,L191x, Q50x, L191F, R25K, F130L, Y27S, D195N, D2G, V20A, V6G, V20I, D2H,P75A, G152D, P74T, C40Y, E8K, Q48x, and any combination thereof.

In some embodiments, a EZH2 mutation is associated with altered histonemethylation patterns. In some embodiments, the EZH2 mutation leads tothe conversion of amino acid Y641 (equivalent to Y646, catalyticdomain), to either F, N, H, S or C resulting in hypertrimethylation ofH3K27 and drives lymphomagenesis. In some embodiments, the EZH2 mutationincludes EZH2 SET-domain mutations, overexpression of EZH2,overexpression of other PRC2 subunits, loss of function mutations ofhistone acetyl transferases (HATs), and loss of function of MLL2. Cellsthat are heterozygous for EZH2 Y646 mutations result inhypertrimethylation of H3K27 relative to cells that are homozygouswild-type (WT) for the EZH2 protein, or to cells that are homozygous forthe Y646 mutation.

In some embodiments, a EZH2 mutation includes, but is not limited to,Y646F, Y646N, D185H, Y646F, Y646S, Y646H, R690H, Y646X, E745K, Y646C,V626M, V679M, R690H, R684H, A682G, E249K, G159R, R288Q, N322S, A692V,R690C, D730* (insertion frameshift), S695L, R684C, M667T, R288*, S644*,D192N, K550T, Q653E, D664G, R347Q, Y646C, G660R, R213C, A255T, S538L,N693K, I55M, R561H, A692V, K515R, Y733*, R63*, Q570*, Q328*, R25Q, T467PA656V, T573I, C571Y, E725K, R16W, P577L, F145S, V680M, G686D, G135R,K634E, S652F, R298C, G648E, R566H, L149R, R502Q, Y731D, R313W, N675K,S652C, T374Hfs*3, N152Ifs*15, E401Kfs*22, K406Mfs*17, E246*, S624C,I146T, V626M, L674S, H694R, A581S, and any combination thereof.

In some embodiments, a JAK2 mutation is a mutation in the JAK2 geneincludes, but is not limited to, T1923C mutation in combination with aG1920T mutation, a G1920T/C1922T mutation, or a G1920A mutation. In someembodiments, the JAK2 mutation is a mutant JAK2 protein comprising oneor more substitutions include, but are not limited to, V617F, V617I,R683G, N542_E543del, E543_D544del, R683S, R683X, F537_K539delinsL(deletion in frame), K539L, N1108S, R1113H, R1063H, R487C, I540Mfs*3(deletion-frameshift), R867Q, K539L, G571S, R1113C, R938Q, R228Q, L830*,E1080*, K539L, C618R, R564Q, D1036H, L1088S, H538Nfs*4, D873N, V392M,I682F, L393V, M535I, C618R, T875N, L611V, D319N, L611S, G921S, H538Y,S1035L, and any combination thereof.

In some embodiments, a FBXW7 mutation is a point mutation selected fromthe group consisting of W244* (*:stop codon), R222*, R278*, E192A,S282*, E113D, R465H/C, 726+1 G>A splice, R505C, R479Q, R465C, R367*,R499Vfs*25 (fs*: frameshift), R658*, D600Y, D520N, D520Y, and anycombination thereof. In further embodiments, the FBXW7 mutation isdouble- or triple-mutation includes, but is not limited to, R479Q andS582L, R465H and S582L, D520N, D520Y and R14Q, and R367* and S582L.

In some embodiments, a CCND3 mutation includes, but is not limited to,S259A, R271Pfs*53 (insertion-caused frameshift), E51*, Q260*, P199S,T283A, T283P, V287D, D286 T288del, R271Gfs*33, Q276*, R241Q, D238G,R33P, 1290K, 1290T, 1290R, P267fs, P284S, P284L, P100S, E253D, S262I,R14W, R114L, D238N, A266E, R167W, and any combination thereof.

In some embodiments, a GNA11 mutation includes, but is not limited to,Q209L, R183C, T257=, R183C, G208Afs*16, Q209H, R183C, Q209P, Q209R,Q209H, ?T96=, R210W, R256Q, T334=, G48D, S53G, Q209P, R213Q, and anycombination thereof. In some embodiments, the GNA11 mutation has twomutations in exon 4, e.g., a mutation in V182 and a mutation in T175, orone or more mutations in exon 5.

2. Combinations with PD-1 and PD-L1 Inhibitors

In some embodiments, the TIL therapy provided to patients with cancermay include treatment with therapeutic populations of TILs alone or mayinclude a combination treatment including TILs and one or more PD-1and/or PD-L1 inhibitors.

Programmed death 1 (PD-1) is a 288-amino acid transmembraneimmunocheckpoint receptor protein expressed by T cells, B cells, naturalkiller (NK) T cells, activated monocytes, and dendritic cells. PD-1,which is also known as CD279, belongs to the CD28 family, and in humansis encoded by the Pdcd1 gene on chromosome 2. PD-1 consists of oneimmunoglobulin (Ig) superfamily domain, a transmembrane region, and anintracellular domain containing an immunoreceptor tyrosine-basedinhibitory motif (ITIM) and an immunoreceptor tyrosine-based switchmotif (ITSM). PD-1 and its ligands (PD-L1 and PD-L2) are known to play akey role in immune tolerance, as described in Keir, et al., Annu. Rev.Immunol. 2008, 26, 677-704. PD-1 provides inhibitory signals thatnegatively regulate T cell immune responses. PD-L1 (also known as B7-H1or CD274) and PD-L2 (also known as B7-DC or CD273) are expressed ontumor cells and stromal cells, which may be encountered by activated Tcells expressing PD-1, leading to immunosuppression of the T cells.PD-L1 is a 290 amino acid transmembrane protein encoded by the Cd274gene on human chromosome 9. Blocking the interaction between PD-1 andits ligands PD-L1 and PD-L2 by use of a PD-1 inhibitor, a PD-L1inhibitor, and/or a PD-L2 inhibitor can overcome immune resistance, asdemonstrated in recent clinical studies, such as that described inTopalian, et al., N. Eng. J. Med. 2012, 366, 2443-54. PD-L1 is expressedon many tumor cell lines, while PD-L2 is expressed is expressed mostlyon dendritic cells and a few tumor lines. In addition to T cells (whichinducibly express PD-1 after activation), PD-1 is also expressed on Bcells, natural killer cells, macrophages, activated monocytes, anddendritic cells.

In an embodiment, the PD-1 inhibitor may be any PD-1 inhibitor or PD-1blocker known in the art. In particular, it is one of the PD-1inhibitors or blockers described in more detail in the followingparagraphs. The terms “inhibitor,” “antagonist,” and “blocker” are usedinterchangeably herein in reference to PD-1 inhibitors. For avoidance ofdoubt, references herein to a PD-1 inhibitor that is an antibody mayrefer to a compound or antigen-binding fragments, variants, conjugates,or biosimilars thereof. For avoidance of doubt, references herein to aPD-1 inhibitor may also refer to a small molecule compound or apharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, orprodrug thereof.

In a preferred embodiment, the PD-1 inhibitor is an antibody (i.e., ananti-PD-1 antibody), a fragment thereof, including Fab fragments, or asingle-chain variable fragment (scFv) thereof. In some embodiments thePD-1 inhibitor is a polyclonal antibody. In a preferred embodiment, thePD-1 inhibitor is a monoclonal antibody. In some embodiments, the PD-1inhibitor competes for binding with PD-1, and/or binds to an epitope onPD-1. In an embodiment, the antibody competes for binding with PD-1,and/or binds to an epitope on PD-1.

In some embodiments, the PD-1 inhibitor is one that binds human PD-1with a KD of about 100 pM or lower, binds human PD-1 with a KD of about90 pM or lower, binds human PD-1 with a KD of about 80 pM or lower,binds human PD-1 with a KD of about 70 pM or lower, binds human PD-1with a KD of about 60 pM or lower, binds human PD-1 with a KD of about50 pM or lower, binds human PD-1 with a KD of about 40 pM or lower,binds human PD-1 with a KD of about 30 pM or lower, binds human PD-1with a KD of about 20 pM or lower, binds human PD-1 with a KD of about10 pM or lower, or binds human PD-1 with a KD of about 1 pM or lower.

In some embodiments, the PD-1 inhibitor is one that binds to human PD-1with a kassoc of about 7.5×105 l/M·s or faster, binds to human PD-1 witha kassoc of about 7.5×105 l/M·s or faster, binds to human PD-1 with akassoc of about 8×105 l/M·s or faster, binds to human PD-1 with a kassocof about 8.5×105 l/M·s or faster, binds to human PD-1 with a kassoc ofabout 9×105 l/M·s or faster, binds to human PD-1 with a kassoc of about9.5×105 l/M·s or faster, or binds to human PD-1 with a kassoc of about1×106 l/M·s or faster.

In some embodiments, the PD-1 inhibitor is one that binds to human PD-1with a kdissoc of about 2×10-5 l/s or slower, binds to human PD-1 with akdissoc of about 2.1×10-5 l/s or slower, binds to human PD-1 with akdissoc of about 2.2×10-5 l/s or slower, binds to human PD-1 with akdissoc of about 2.3×10-5 l/s or slower, binds to human PD-1 with akdissoc of about 2.4×10-5 l/s or slower, binds to human PD-1 with akdissoc of about 2.5×10-5 l/s or slower, binds to human PD-1 with akdissoc of about 2.6×10-5 l/s or slower or binds to human PD-1 with akdissoc of about 2.7×10-5 l/s or slower, binds to human PD-1 with akdissoc of about 2.8×10-5 l/s or slower, binds to human PD-1 with akdissoc of about 2.9×10-5 l/s or slower, or binds to human PD-1 with akdissoc of about 3×10-5 l/s or slower.

In some embodiments, the PD-1 inhibitor is one that blocks or inhibitsbinding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 ofabout 10 nM or lower, blocks or inhibits binding of human PD-L1 or humanPD-L2 to human PD-1 with an IC50 of about 9 nM or lower, blocks orinhibits binding of human PD-L1 or human PD-L2 to human PD-1 with anIC50 of about 8 nM or lower, blocks or inhibits binding of human PD-L1or human PD-L2 to human PD-1 with an IC50 of about 7 nM or lower, blocksor inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with anIC50 of about 6 nM or lower, blocks or inhibits binding of human PD-L1or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower, blocksor inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with anIC50 of about 4 nM or lower, blocks or inhibits binding of human PD-L1or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower, blocksor inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with anIC50 of about 2 nM or lower, or blocks or inhibits binding of humanPD-L1 or human PD-L2 to human PD-1 with an IC50 of about 1 nM or lower.

In an embodiment, the PD-1 inhibitor is nivolumab (commerciallyavailable as OPDIVO from Bristol-Myers Squibb Co.), or biosimilars,antigen-binding fragments, conjugates, or variants thereof. Nivolumab isa fully human IgG4 antibody blocking the PD-1 receptor. In anembodiment, the anti-PD-1 antibody is an immunoglobulin G4 kappa,anti-(human CD274) antibody. Nivolumab is assigned Chemical AbstractsService (CAS) registry number 946414-94-4 and is also known as 5C4,BMS-936558, MDX-1106, and ONO-4538. The preparation and properties ofnivolumab are described in U.S. Pat. No. 8,008,449 and InternationalPatent Publication No. WO 2006/121168, the disclosures of which areincorporated by reference herein. The clinical safety and efficacy ofnivolumab in various forms of cancer has been described in Wang, et al.,Cancer Immunol. Res. 2014, 2, 846-56; Page, et al., Ann. Rev. Med.,2014, 65, 185-202; and Weber, et al., J. Clin. Oncology, 2013, 31,4311-4318, the disclosures of which are incorporated by referenceherein. The amino acid sequences of nivolumab are set forth in Table 18.Nivolumab has intra-heavy chain disulfide linkages at 22-96,140-196,254-314, 360-418, 22″-96″, 140″-196″, 254″-314″, and 360″-418″;intra-light chain disulfide linkages at 23′-88′, 134′-194′, 23′″-88″,and 134′″-194′″; inter-heavy-light chain disulfide linkages at 127-214′,127″-214′″, inter-heavy-heavy chain disulfide linkages at 219-219″ and222-222″; and N-glycosylation sites (H CH2 84.4) at 290, 290″.

In an embodiment, a PD-1 inhibitor comprises a heavy chain given by SEQID NO:158 and a light chain given by SEQ ID NO:159. In an embodiment, aPD-1 inhibitor comprises heavy and light chains having the sequencesshown in SEQ ID NO:158 and SEQ ID NO:159, respectively, or antigenbinding fragments, Fab fragments, single-chain variable fragments(scFv), variants, or conjugates thereof. In an embodiment, a PD-1inhibitor comprises heavy and light chains that are each at least 99%identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159,respectively. In an embodiment, a PD-1 inhibitor comprises heavy andlight chains that are each at least 98% identical to the sequences shownin SEQ ID NO:158 and SEQ ID NO:159, respectively. In an embodiment, aPD-1 inhibitor comprises heavy and light chains that are each at least97% identical to the sequences shown in SEQ ID NO:158 and SEQ ID NO:159,respectively. In an embodiment, a PD-1 inhibitor comprises heavy andlight chains that are each at least 96% identical to the sequences shownin SEQ ID NO:158 and SEQ ID NO:159, respectively. In an embodiment, aPD-1 inhibitor comprises heavy and light chains that are each at least95% identical to the sequences shown in SEQ ID NO:463 and SEQ ID NO:159,respectively.

In an embodiment, the PD-1 inhibitor comprises the heavy and light chainCDRs or variable regions (VRs) of nivolumab. In an embodiment, the PD-1inhibitor heavy chain variable region (VH) comprises the sequence shownin SEQ ID NO:160, and the PD-1 inhibitor light chain variable region(VL) comprises the sequence shown in SEQ ID NO:161, or conservativeamino acid substitutions thereof. In an embodiment, a PD-1 inhibitorcomprises VH and VL regions that are each at least 99% identical to thesequences shown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In anembodiment, a PD-1 inhibitor comprises VH and VL regions that are eachat least 98% identical to the sequences shown in SEQ ID NO:160 and SEQID NO:161, respectively. In an embodiment, a PD-1 inhibitor comprises VHand VL regions that are each at least 97% identical to the sequencesshown in SEQ ID NO:160 and SEQ ID NO:161, respectively. In anembodiment, a PD-1 inhibitor comprises VH and VL regions that are eachat least 96% identical to the sequences shown in SEQ ID NO:160 and SEQID NO:161, respectively. In an embodiment, a PD-1 inhibitor comprises VHand VL regions that are each at least 95% identical to the sequencesshown in SEQ ID NO:160 and SEQ ID NO:161, respectively.

In an embodiment, a PD-1 inhibitor comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:162, SEQ IDNO:163, and SEQ ID NO:164, respectively, or conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:165, SEQ ID NO:166, and SEQID NO:167, respectively, or conservative amino acid substitutionsthereof. In an embodiment, the antibody competes for binding with,and/or binds to the same epitope on PD-1 as any of the aforementionedantibodies.

In an embodiment, the PD-1 inhibitor is an anti-PD-1 biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to nivolumab. In an embodiment, the biosimilar comprises ananti-PD-1 antibody comprising an amino acid sequence which has at least97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, tothe amino acid sequence of a reference medicinal product or referencebiological product and which comprises one or more post-translationalmodifications as compared to the reference medicinal product orreference biological product, wherein the reference medicinal product orreference biological product is nivolumab. In some embodiments, the oneor more post-translational modifications are selected from one or moreof: glycosylation, oxidation, deamidation, and truncation. In someembodiments, the biosimilar is an anti-PD-1 antibody authorized orsubmitted for authorization, wherein the anti-PD-1 antibody is providedin a formulation which differs from the formulations of a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is nivolumab. Theanti-PD-1 antibody may be authorized by a drug regulatory authority suchas the U.S. FDA and/or the European Union's EMA. In some embodiments,the biosimilar is provided as a composition which further comprises oneor more excipients, wherein the one or more excipients are the same ordifferent to the excipients comprised in a reference medicinal productor reference biological product, wherein the reference medicinal productor reference biological product is nivolumab. In some embodiments, thebiosimilar is provided as a composition which further comprises one ormore excipients, wherein the one or more excipients are the same ordifferent to the excipients comprised in a reference medicinal productor reference biological product, wherein the reference medicinal productor reference biological product is nivolumab.

TABLE 18 Amino acid sequences for PD-1 inhibitors related to nivolumab.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 463QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY  60nivolumabADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS 120heavy chainVFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS 160VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKP 240KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT 300VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC 360LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV 420MHEALHNHYT QKSLSLSLGK 440 SEQ ID NO: 159EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA  60nivolumabRFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP 120light chainSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ ID NO: 160QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY  60nivolumab ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSS 113variable heavy chain SEQ ID NO: 161EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA  60nivolumab RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIK 107variable light chain SEQ ID NO: 162 NSGMH   5 nivolumab heavy chain CDR1SEQ ID NO: 163 VIWYDGSKRY YADSVKG  17 nivolumab heavy chain CDR2SEQ ID NO: 164 NDDY   4 nivolumab heavy chain CDR3 SEQ ID NO: 165RASQSVSSYL A  11 nivolumab light chain CDR1 SEQ ID NO: 166 DASNRAT   7nivolumab light chain CDR2 SEQ ID NO: 167 QQSSNWPRT   9 nivolumablight chain CDR3

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilarthereof, and the nivolumab is administered at a dose of about 0.5 mg/kgto about 10 mg/kg. In some embodiments, the PD-1 inhibitor is nivolumabor a biosimilar thereof, and the nivolumab is administered at a dose ofabout 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg,about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, thenivolumab administration is begun 1, 2, 3, 4, or 5 days post IL-2administration. In some embodiments, the nivolumab administration isbegun 1, 2, or 3 days post IL-2 administration. In some embodiments, thenivolumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection(i.e., before obtaining a tumor sample from the subject or patient). Insome embodiments, the nivolumab can also be administered 1, 2, or 3weeks pre-resection (i.e., before obtaining a tumor sample from thesubject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilarthereof, and the nivolumab is administered at a dose of about 200 mg toabout 500 mg. In some embodiments, the PD-1 inhibitor is nivolumab or abiosimilar thereof, and the nivolumab is administered at a dose of about200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about500 mg. In some embodiments, the nivolumab administration is begun 1, 2,3, 4, or 5 days post IL-2 administration. In some embodiments, thenivolumab administration is begun 1, 2, or 3 days post IL-2administration. In some embodiments, the nivolumab can also beadministered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., before obtaininga tumor sample from the subject or patient). In some embodiments, thenivolumab can also be administered 1, 2, or 3 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilarthereof, and the nivolumab is administered every 2 weeks, every 3 weeks,every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, thenivolumab administration is begun 1, 2, 3, 4, or 5 days post IL-2administration. In some embodiments, the nivolumab administration isbegun 1, 2, or 3 days post IL-2 administration. In some embodiments, thenivolumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection(i.e., before obtaining a tumor sample from the subject or patient). Insome embodiments, the nivolumab can also be administered 1, 2, or 3weeks pre-resection (i.e., before obtaining a tumor sample from thesubject or patient).

In some embodiments, the nivolumab is administered to treat unresectableor metastatic melanoma. In some embodiments, the nivolumab isadministered to treat unresectable or metastatic melanoma and isadministered at about 240 mg every 2 weeks. In some embodiments, thenivolumab is administered to treat unresectable or metastatic melanomaand is administered at about 480 mg every 4 weeks. In some embodiments,the nivolumab is administered to treat unresectable or metastaticmelanoma and is administered at about 1 mg/kg followed by ipilimumab 3mg/kg on the same day every 3 weeks for 4 doses, then 240 mg every 2weeks or 480 mg every 4 weeks. In some embodiments, the nivolumabadministration is begun 1, 2, 3, 4, or 5 days post IL-2 administration.In some embodiments, the nivolumab administration is begun 1, 2, or 3days post IL-2 administration. In some embodiments, the nivolumab canalso be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., beforeobtaining a tumor sample from the subject or patient). In someembodiments, the nivolumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In some embodiments, the nivolumab is administered for the adjuvanttreatment of melanoma. In some embodiments, the nivolumab isadministered for the adjuvant treatment of melanoma at about 240 mgevery 2 weeks. In some embodiments, the nivolumab is administered forthe adjuvant treatment of melanoma at about 480 mg every 4 weeks. Insome embodiments, the nivolumab administration is begun 1, 2, 3, 4, or 5days post IL-2 administration. In some embodiments, the nivolumabadministration is begun 1, 2, or 3 days post IL-2 administration. Insome embodiments, the nivolumab can also be administered 1, 2, 3, 4 or 5weeks pre-resection (i.e., before obtaining a tumor sample from thesubject or patient). In some embodiments, the nivolumab can also beadministered 1, 2, or 3 weeks pre-resection (i.e., before obtaining atumor sample from the subject or patient).

In some embodiments, the nivolumab is administered to treat metastaticnon-small cell lung cancer. In some embodiments, the nivolumab isadministered to treat metastatic non-small cell lung cancer at about 3mg/kg every 2 weeks along with ipilimumab at about 1 mg/kg every 6weeks. In some embodiments, the nivolumab is administered to treatmetastatic non-small cell lung cancer at about 360 mg every 3 weeks withipilimumab 1 mg/kg every 6 weeks and 2 cycles of platinum-doubletchemotherapy. In some embodiments, the nivolumab is administered totreat metastatic non-small cell lung cancer at about 240 mg every 2weeks or 480 mg every 4 weeks. In some embodiments, the nivolumabadministration is begun 1, 2, 3, 4, or 5 days post IL-2 administration.In some embodiments, the nivolumab administration is begun 1, 2, or 3days post IL-2 administration. In some embodiments, the nivolumab canalso be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., beforeobtaining a tumor sample from the subject or patient). In someembodiments, the nivolumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In some embodiments, the nivolumab is administered to treat small celllung cancer. In some embodiments, the nivolumab is administered to treatsmall cell lung cancer at about 240 mg every 2 weeks. In someembodiments, the nivolumab administration is begun 1, 2, 3, 4, or 5 dayspost IL-2 administration. In some embodiments, the nivolumabadministration is begun 1, 2, or 3 days post IL-2 administration. Insome embodiments, the nivolumab can also be administered 1, 2, 3, 4 or 5weeks pre-resection (i.e., before obtaining a tumor sample from thesubject or patient). In some embodiments, the nivolumab can also beadministered 1, 2, or 3 weeks pre-resection (i.e., before obtaining atumor sample from the subject or patient).

In some embodiments, the nivolumab is administered to treat malignantpleural mesothelioma at about 360 mg every 3 weeks with ipilimumab 1mg/kg every 6 weeks. In some embodiments, the nivolumab administrationis begun 1, 2, 3, 4, or 5 days post IL-2 administration. In someembodiments, the nivolumab administration is begun 1, 2, or 3 days postIL-2 administration. In some embodiments, the nivolumab can also beadministered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., before obtaininga tumor sample from the subject or patient). In some embodiments, thenivolumab can also be administered 1, 2, or 3 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient).

In some embodiments, the nivolumab is administered to treat advancedrenal cell carcinoma. In some embodiments, the nivolumab is administeredto treat advanced renal cell carcinoma at about 240 mg every 2 weeks. Insome embodiments, the nivolumab is administered to treat advanced renalcell carcinoma at about 480 mg every 4 weeks. In some embodiments, thenivolumab is administered to treat advanced renal cell carcinoma atabout 3 mg/kg followed by ipilimumab at about 1 mg/kg on the same dayevery 3 weeks for 4 doses, then 240 mg every 2 weeks. In someembodiments, the nivolumab is administered to treat advanced renal cellcarcinoma at about 3 mg/kg followed by ipilimumab at about 1 mg/kg onthe same day every 3 weeks for 4 doses, then 240 mg every 2 weeks 480 mgevery 4 weeks. In some embodiments, the nivolumab administration isbegun 1, 2, 3, 4, or 5 days post IL-2 administration. In someembodiments, the nivolumab administration is begun 1, 2, or 3 days postIL-2 administration. In some embodiments, the nivolumab can also beadministered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., before obtaininga tumor sample from the subject or patient). In some embodiments, thenivolumab can also be administered 1, 2, or 3 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient).

In some embodiments, the nivolumab is administered to treat classicalHodgkin lymphoma. In some embodiments, the nivolumab is administered totreat classical Hodgkin lymphoma at about 240 mg every 2 weeks. In someembodiments, the nivolumab is administered to treat classical Hodgkinlymphoma at about 480 mg every 4 weeks. In some embodiments, thenivolumab administration is begun 1, 2, 3, 4, or 5 days post IL-2administration. In some embodiments, the nivolumab administration isbegun 1, 2, or 3 days post IL-2 administration. In some embodiments, thenivolumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection(i.e., before obtaining a tumor sample from the subject or patient). Insome embodiments, the nivolumab can also be administered 1, 2, or 3weeks pre-resection (i.e., before obtaining a tumor sample from thesubject or patient).

In some embodiments, the nivolumab is administered to treat Recurrent ormetastatic squamous cell carcinoma of the head and neck. In someembodiments, the nivolumab is administered to treat recurrent ormetastatic squamous cell carcinoma of the head and neck at about 240 mgevery 2 weeks. In some embodiments, the nivolumab is administered totreat recurrent or metastatic squamous cell carcinoma of the head andneck at about 480 mg every 4 weeks. In some embodiments, the nivolumabadministration is begun 1, 2, 3, 4, or 5 days post IL-2 administration.In some embodiments, the nivolumab administration is begun 1, 2, or 3days post IL-2 administration. In some embodiments, the nivolumab canalso be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., beforeobtaining a tumor sample from the subject or patient). In someembodiments, the nivolumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In some embodiments, the nivolumab is administered to treat locallyadvanced or metastatic urothelial carcinoma at about 240 mg every 2weeks. In some embodiments, the nivolumab is administered to treatlocally advanced or metastatic urothelial carcinoma at about 480 mgevery 4 weeks. In some embodiments, the nivolumab administration isbegun 1, 2, 3, 4, or 5 days post IL-2 administration. In someembodiments, the nivolumab administration is begun 1, 2, or 3 days postIL-2 administration. In some embodiments, the nivolumab can also beadministered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., before obtaininga tumor sample from the subject or patient). In some embodiments, thenivolumab can also be administered 1, 2, or 3 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient).

In some embodiments, the nivolumab is administered to treatmicrosatellite instability-high (MSI-H) or mismatch repair deficient(dMMR) metastatic colorectal cancer. In some embodiments, the nivolumabis administered to treat microsatellite instability-high (MSI-H) ormismatch repair deficient (dMMR) metastatic colorectal cancer in adultand pediatric patients. In some embodiments, the nivolumab isadministered to treat microsatellite instability-high (MSI-H) ormismatch repair deficient (dMMR) metastatic colorectal cancer in adultand pediatric patients >40 kg at about 240 mg every 2 weeks. In someembodiments, the nivolumab is administered to treat microsatelliteinstability-high (MSI-H) or mismatch repair deficient (dMMR) metastaticcolorectal cancer in adult and pediatric patients >40 kg at about 480 mgevery 4 weeks. In some embodiments, the nivolumab administration isbegun 1, 2, 3, 4, or 5 days post IL-2 administration. In someembodiments, the nivolumab administration is begun 1, 2, or 3 days postIL-2 administration. In some embodiments, the nivolumab can also beadministered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., before obtaininga tumor sample from the subject or patient). In some embodiments, thenivolumab can also be administered 1, 2, or 3 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient).

In some embodiments, the nivolumab is administered to treatmicrosatellite instability-high (MSI-H) or mismatch repair deficient(dMMR) metastatic colorectal cancer in pediatric patients <40 kg atabout 3 mg/kg every 2 weeks. In some embodiments, the nivolumab isadministered to treat microsatellite instability-high (MSI-H) ormismatch repair deficient (dMMR) metastatic colorectal cancer in adultand pediatric patients >40 kg at about 3 mg/kg followed by ipilimumab 1mg/kg on the same day every 3 weeks for 4 doses, then 240 mg every 2weeks. In some embodiments, the nivolumab is administered to treatmicrosatellite instability-high (MSI-H) or mismatch repair deficient(dMMR) metastatic colorectal cancer in adult and pediatric patients >40kg at about 3 mg/kg followed by ipilimumab 1 mg/kg on the same day every3 weeks for 4 doses, then 480 mg every 4 weeks. In some embodiments, thenivolumab administration is begun 1, 2, 3, 4, or 5 days post IL-2administration. In some embodiments, the nivolumab administration isbegun 1, 2, or 3 days post IL-2 administration. In some embodiments, thenivolumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection(i.e., before obtaining a tumor sample from the subject or patient). Insome embodiments, the nivolumab can also be administered 1, 2, or 3weeks pre-resection (i.e., before obtaining a tumor sample from thesubject or patient).

In some embodiments, the nivolumab is administered to treathepatocellular carcinoma. In some embodiments, the nivolumab isadministered to treat hepatocellular carcinoma at about 240 mg every 2weeks. In some embodiments, the nivolumab is administered to treathepatocellular carcinoma at about 480 mg every 4 weeks. In someembodiments, the nivolumab is administered to treat hepatocellularcarcinoma at about 1 mg/kg followed by ipilimumab 3 mg/kg on the sameday every 3 weeks for 4 doses, then 240 mg every 2 weeks. In someembodiments, the nivolumab is administered to treat hepatocellularcarcinoma at about 1 mg/kg followed by ipilimumab 3 mg/kg on the sameday every 3 weeks for 4 doses, then 480 mg every 4 weeks. In someembodiments, the nivolumab administration is begun 1, 2, 3, 4, or 5 dayspost IL-2 administration. In some embodiments, the nivolumabadministration is begun 1, 2, or 3 days post IL-2 administration. Insome embodiments, the nivolumab can also be administered 1, 2, 3, 4 or 5weeks pre-resection (i.e., before obtaining a tumor sample from thesubject or patient). In some embodiments, the nivolumab can also beadministered 1, 2, or 3 weeks pre-resection (i.e., before obtaining atumor sample from the subject or patient).

In some embodiments, the nivolumab is administered to treat esophagealsquamous cell carcinoma. In some embodiments, the nivolumab isadministered to treat esophageal squamous cell carcinoma at about 240 mgevery 2 weeks. In some embodiments, the nivolumab is administered totreat esophageal squamous cell carcinoma at about 480 mg every 4 weeks.In some embodiments, the nivolumab administration is begun 1, 2, 3, 4,or 5 days post IL-2 administration. In some embodiments, the nivolumabadministration is begun 1, 2, or 3 days post IL-2 administration. Insome embodiments, the nivolumab can also be administered 1, 2, 3, 4 or 5weeks pre-resection (i.e., before obtaining a tumor sample from thesubject or patient). In some embodiments, the nivolumab can also beadministered 1, 2, or 3 weeks pre-resection (i.e., before obtaining atumor sample from the subject or patient).

In another embodiment, the PD-1 inhibitor comprises pembrolizumab(commercially available as KEYTRUDA from Merck & Co., Inc., Kenilworth,N.J., USA), or antigen-binding fragments, conjugates, or variantsthereof. Pembrolizumab is assigned CAS registry number 1374853-91-4 andis also known as lambrolizumab, MK-3475, and SCH-900475. Pembrolizumabhas an immunoglobulin G4, anti-(human protein PDCD1 (programmed celldeath 1)) (human-Mus musculus monoclonal heavy chain), disulfide withhuman-Mus musculus monoclonal light chain, dimer structure. Thestructure of pembrolizumab may also be described as immunoglobulin G4,anti-(human programmed cell death 1); humanized mouse monoclonal[228-L-proline(H10-S>P)]γ4 heavy chain (134-218′)-disulfide withhumanized mouse monoclonal κ light chain dimer (226-226″:229-229″)-bisdisulfide. The properties, uses, and preparation ofpembrolizumab are described in International Patent Publication No. WO2008/156712 A1, U.S. Pat. No. 8,354,509 and U.S. Patent ApplicationPublication Nos. US 2010/0266617 A1, US 2013/0108651 A1, and US2013/0109843 A2, the disclosures of which are incorporated herein byreference. The clinical safety and efficacy of pembrolizumab in variousforms of cancer is described in Fuerst, Oncology Times, 2014, 36, 35-36;Robert, et al., Lancet, 2014, 384, 1109-17; and Thomas, et al., Exp.Opin. Biol. Ther., 2014, 14, 1061-1064. The amino acid sequences ofpembrolizumab are set forth in Table 19. Pembrolizumab includes thefollowing disulfide bridges: 22-96, 22″-96″, 23′-92′, 23′″-92″,134-218′, 134″-218′″, 138′-198′, 138′″-198′″, 147-203, 147″-203″,226-226″, 229-229″, 261-321, 261″-321″, 367-425, and 367″-425″, and thefollowing glycosylation sites (N): Asn-297 and Asn-297″. Pembrolizumabis an IgG4/kappa isotype with a stabilizing S228P mutation in the Fcregion; insertion of this mutation in the IgG4 hinge region prevents theformation of half molecules typically observed for IgG4 antibodies.Pembrolizumab is heterogeneously glycosylated at Asn297 within the Fcdomain of each heavy chain, yielding a molecular weight of approximately149 kDa for the intact antibody. The dominant glycoform of pembrolizumabis the fucosylated agalacto diantennary glycan form (G0F).

In an embodiment, a PD-1 inhibitor comprises a heavy chain given by SEQID NO:168 and a light chain given by SEQ ID NO:169. In an embodiment, aPD-1 inhibitor comprises heavy and light chains having the sequencesshown in SEQ ID NO:168 and SEQ ID NO:169, respectively, or antigenbinding fragments, Fab fragments, single-chain variable fragments(scFv), variants, or conjugates thereof. In an embodiment, a PD-1inhibitor comprises heavy and light chains that are each at least 99%identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169,respectively. In an embodiment, a PD-1 inhibitor comprises heavy andlight chains that are each at least 98% identical to the sequences shownin SEQ ID NO:168 and SEQ ID NO:169, respectively. In an embodiment, aPD-1 inhibitor comprises heavy and light chains that are each at least97% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169,respectively. In an embodiment, a PD-1 inhibitor comprises heavy andlight chains that are each at least 96% identical to the sequences shownin SEQ ID NO:168 and SEQ ID NO:169, respectively. In an embodiment, aPD-1 inhibitor comprises heavy and light chains that are each at least95% identical to the sequences shown in SEQ ID NO:168 and SEQ ID NO:169,respectively.

In an embodiment, the PD-1 inhibitor comprises the heavy and light chainCDRs or variable regions (VRs) of pembrolizumab. In an embodiment, thePD-1 inhibitor heavy chain variable region (V_(H)) comprises thesequence shown in SEQ ID NO:170, and the PD-1 inhibitor light chainvariable region (V_(L)) comprises the sequence shown in SEQ ID NO:171,or conservative amino acid substitutions thereof. In an embodiment, aPD-1 inhibitor comprises V_(H) and V_(L) regions that are each at least99% identical to the sequences shown in SEQ ID NO:170 and SEQ ID NO:171,respectively. In an embodiment, a PD-1 inhibitor comprises V_(H) andV_(L) regions that are each at least 98% identical to the sequencesshown in SEQ ID NO:170 and SEQ ID NO:171, respectively. In anembodiment, a PD-1 inhibitor comprises V_(H) and V_(L) regions that areeach at least 97% identical to the sequences shown in SEQ ID NO:170 andSEQ ID NO:171, respectively. In an embodiment, a PD-1 inhibitorcomprises V_(H) and V_(L) regions that are each at least 96% identicalto the sequences shown in SEQ ID NO:170 and SEQ ID NO:171, respectively.In an embodiment, a PD-1 inhibitor comprises V_(H) and V_(L) regionsthat are each at least 95% identical to the sequences shown in SEQ IDNO:170 and SEQ ID NO:171, respectively.

In an embodiment, a PD-1 inhibitor comprises the heavy chain CDR1, CDR2and CDR3 domains having the sequences set forth in SEQ ID NO:172, SEQ IDNO:173, and SEQ ID NO:174, respectively, or conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:175, SEQ ID NO:176, and SEQID NO:177, respectively, or conservative amino acid substitutionsthereof. In an embodiment, the antibody competes for binding with,and/or binds to the same epitope on PD-1 as any of the aforementionedantibodies.

In an embodiment, the PD-1 inhibitor is an anti-PD-1 biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to pembrolizumab. In an embodiment, the biosimilar comprisesan anti-PD-1 antibody comprising an amino acid sequence which has atleast 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequenceidentity, to the amino acid sequence of a reference medicinal product orreference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is pembrolizumab. In someembodiments, the one or more post-translational modifications areselected from one or more of: glycosylation, oxidation, deamidation, andtruncation. In some embodiments, the biosimilar is an anti-PD-1 antibodyauthorized or submitted for authorization, wherein the anti-PD-1antibody is provided in a formulation which differs from theformulations of a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is pembrolizumab. The anti-PD-1 antibody may be authorized by adrug regulatory authority such as the U.S. FDA and/or the EuropeanUnion's EMA. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is pembrolizumab. In some embodiments, the biosimilar isprovided as a composition which further comprises one or moreexcipients, wherein the one or more excipients are the same or differentto the excipients comprised in a reference medicinal product orreference biological product, wherein the reference medicinal product orreference biological product is pembrolizumab.

TABLE 19Amino acid sequences for PD-1 inhibitors related to pembrolizumab.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 168QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF  60pembrolizumabNEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS 120heavy chainASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 180GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV 240FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY 300RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK 360NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG 420NVFSCSVMHE ALHNHYTQKS LSLSLGK 447 SEQ ID NO: 169EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL LIYLASYLES  60pembrolizumabGVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEI KRTVAAPSVF 120light chainIFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 180STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 210 SEQ ID NO: 170QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF  60pembrolizumabNEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS 120variable heavy chain SEQ ID NO: 171EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL LIYLASYLES  60pembrolizumab GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEI K111 variable light chain SEQ ID NO: 172 NYYMY   5 pembrolizumabheavy chain CDR1 SEQ ID NO: 173 GINPSNGGTN FNEKFK  16 pembrolizumabheavy chain CDR2 SEQ ID NO: 174 RDYRFDMGFD Y  11 pembrolizumabheavy chain CDR3 SEQ ID NO: 175 RASKGVSTSG YSYLH  15 pembrolizumablight chain CDR1 SEQ ID NO: 176 LASYLES   7 pembrolizumab light chainCDR2 SEQ ID NO: 177 QHSRDLPLT   9 pembrolizumab light chain CDR3

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilarthereof, and the pembrolizumab is administered at a dose of about 0.5mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor ispembrolizumab or a biosimilar thereof, and the pembrolizumab isadministered at a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg,about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg,about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. Insome embodiments, the pembrolizumab administration is begun 1, 2, 3, 4,or 5 days post IL-2 administration. In some embodiments, thepembrolizumab administration is begun 1, 2, or 3 days post IL-2administration. In some embodiments, the pembrolizumab can also beadministered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., before obtaininga tumor sample from the subject or patient). In some embodiments, thepembrolizumab can also be administered 1, 2, or 3 weeks pre-resection(i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilarthereof, wherein the pembrolizumab is administered at a dose of about200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor ispembrolizumab or a biosimilar thereof, and the nivolumab is administeredat a dose of about 200 mg, about 220 mg, about 240 mg, about 260 mg,about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg,about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg,about 480 mg, or about 500 mg. In some embodiments, the pembrolizumabadministration is begun 1, 2, 3, 4, or 5 days post IL-2 administration.In some embodiments, the pembrolizumab administration is begun 1, 2, or3 days post IL-2 administration. In some embodiments, the pembrolizumabcan also be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilarthereof, wherein the pembrolizumab is administered every 2 weeks, every3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In someembodiments, the pembrolizumab administration is begun 1, 2, 3, 4, or 5days post IL-2 administration. In some embodiments, the pembrolizumabadministration is begun 1, 2, or 3 days post IL-2 administration. Insome embodiments, the pembrolizumab can also be administered 1, 2, 3, 4or 5 weeks pre-resection (i.e., before obtaining a tumor sample from thesubject or patient). In some embodiments, the pembrolizumab can also beadministered 1, 2, or 3 weeks pre-resection (i.e., before obtaining atumor sample from the subject or patient).

In some embodiments, the pembrolizumab is administered to treatmelanoma. In some embodiments, the pembrolizumab is administered totreat melanoma at about 200 mg every 3 weeks. In some embodiments, thepembrolizumab is administered to treat melanoma at about 400 mg every 6weeks. In some embodiments, the pembrolizumab administration is begun 1,2, 3, 4, or 5 days post IL-2 administration. In some embodiments, thepembrolizumab administration is begun 1, 2, or 3 days post IL-2administration. In some embodiments, the pembrolizumab can also beadministered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., before obtaininga tumor sample from the subject or patient). In some embodiments, thepembrolizumab can also be administered 1, 2, or 3 weeks pre-resection(i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the pembrolizumab is administered to treat NSCLC.In some embodiments, the pembrolizumab is administered to treat NSCLC atabout 200 mg every 3 weeks. In some embodiments, the pembrolizumab isadministered to treat NSCLC at about 400 mg every 6 weeks. In someembodiments, the pembrolizumab administration is begun 1, 2, 3, 4, or 5days post IL-2 administration. In some embodiments, the pembrolizumabadministration is begun 1, 2, or 3 days post IL-2 administration. Insome embodiments, the pembrolizumab can also be administered 1, 2, 3, 4or 5 weeks pre-resection (i.e., before obtaining a tumor sample from thesubject or patient). In some embodiments, the pembrolizumab can also beadministered 1, 2, or 3 weeks pre-resection (i.e., before obtaining atumor sample from the subject or patient).

In some embodiments, the pembrolizumab is administered to treat smallcell lung cancer (SCLC). In some embodiments, the pembrolizumab isadministered to treat SCLC at about 200 mg every 3 weeks. In someembodiments, the pembrolizumab is administered to treat SCLC at about400 mg every 6 weeks. In some embodiments, the pembrolizumabadministration is begun 1, 2, 3, 4, or 5 days post IL-2 administration.In some embodiments, the pembrolizumab administration is begun 1, 2, or3 days post IL-2 administration. In some embodiments, the pembrolizumabcan also be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In some embodiments, the pembrolizumab is administered to treat head andneck squamous cell cancer (HNSCC). In some embodiments, thepembrolizumab is administered to treat HNSCC at about 200 mg every 3weeks. In some embodiments, the pembrolizumab is administered to treatHNSCCat about 400 mg every 6 weeks. In some embodiments, thepembrolizumab administration is begun 1, 2, 3, 4, or 5 days post IL-2administration. In some embodiments, the pembrolizumab administration isbegun 1, 2, or 3 days post IL-2 administration. In some embodiments, thepembrolizumab can also be administered 1, 2, 3, 4 or 5 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient). In some embodiments, the pembrolizumab can also beadministered 1, 2, or 3 weeks pre-resection (i.e., before obtaining atumor sample from the subject or patient).

In some embodiments, the pembrolizumab is administered to treatclassical Hodgkin lymphoma (cHL) or primary mediastinal large B-celllymphoma (PMBCL) at about 200 mg every 3 weeks. In some embodiments, thepembrolizumab is administered to treat classical Hodgkin lymphoma (cHL)or primary mediastinal large B-cell lymphoma (PMBCL) at about 400 mgevery 6 weeks for adults. In some embodiments, the pembrolizumab isadministered to treat classical Hodgkin lymphoma (cHL) or primarymediastinal large B-cell lymphoma (PMBCL) at about 2 mg/kg (up to 200mg) every 3 weeks for pediatrics. In some embodiments, the pembrolizumabadministration is begun 1, 2, 3, 4, or 5 days post IL-2 administration.In some embodiments, the pembrolizumab administration is begun 1, 2, or3 days post IL-2 administration. In some embodiments, the pembrolizumabcan also be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In some embodiments, the pembrolizumab is administered to treaturothelial carcinoma at about 200 mg every 3 weeks. In some embodiments,the pembrolizumab is administered to treat urothelial carcinoma at about400 mg every 6 weeks. In some embodiments, the pembrolizumabadministration is begun 1, 2, 3, 4, or 5 days post IL-2 administration.In some embodiments, the pembrolizumab administration is begun 1, 2, or3 days post IL-2 administration. In some embodiments, the pembrolizumabcan also be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In some embodiments, the pembrolizumab is administered to treatmicrosatellite instability-high (MSI-H) or mismatch repair deficient(dMMR) cancer at about 200 mg every 3 weeks. In some embodiments, thepembrolizumab is administered to treat MSI-H or dMMR cancer at about 400mg every 6 weeks for adults. In some embodiments, the pembrolizumab isadministered to treat MSI-H or dMMR cancer at about 2 mg/kg (up to 200mg) every 3 weeks for pediatrics. In some embodiments, the pembrolizumabadministration is begun 1, 2, 3, 4, or 5 days post IL-2 administration.In some embodiments, the pembrolizumab administration is begun 1, 2, or3 days post IL-2 administration. In some embodiments, the pembrolizumabcan also be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient). In some embodiments, the pembrolizumab administration is begun1, 2, 3, 4, or 5 days post IL-2 administration. In some embodiments, thepembrolizumab administration is begun 1, 2, or 3 days post IL-2administration. In some embodiments, the pembrolizumab can also beadministered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., before obtaininga tumor sample from the subject or patient). In some embodiments, thepembrolizumab can also be administered 1, 2, or 3 weeks pre-resection(i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the pembrolizumab is administered to treatmicrosatellite instability-high (MSI-H) or mismatch repair deficientcolorectal cancer (dMMR CRC at about 200 mg every 3 weeks. In someembodiments, the pembrolizumab is administered to treat MSI-H or dMMRCRC at about 400 mg every 6 weeks. In some embodiments, thepembrolizumab administration is begun 1, 2, 3, 4, or 5 days post IL-2administration. In some embodiments, the pembrolizumab administration isbegun 1, 2, or 3 days post IL-2 administration. In some embodiments, thepembrolizumab can also be administered 1, 2, 3, 4 or 5 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient). In some embodiments, the pembrolizumab can also beadministered 1, 2, or 3 weeks pre-resection (i.e., before obtaining atumor sample from the subject or patient).

In some embodiments, the pembrolizumab is administered to treat gastriccancer at about 200 mg every 3 weeks. In some embodiments, thepembrolizumab is administered to treat gastric cancer at about 400 mgevery 6 weeks. In some embodiments, the pembrolizumab administration isbegun 1, 2, 3, 4, or 5 days post IL-2 administration. In someembodiments, the pembrolizumab administration is begun 1, 2, or 3 dayspost IL-2 administration. In some embodiments, the pembrolizumab canalso be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., beforeobtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In some embodiments, the pembrolizumab is administered to treatEsophageal Cancer at about 200 mg every 3 weeks. In some embodiments,the pembrolizumab is administered to treat Esophageal Cancer at about400 mg every 6 weeks. In some embodiments, the pembrolizumabadministration is begun 1, 2, 3, 4, or 5 days post IL-2 administration.In some embodiments, the pembrolizumab administration is begun 1, 2, or3 days post IL-2 administration. In some embodiments, the pembrolizumabcan also be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In some embodiments, the pembrolizumab is administered to treat cervicalcancer at about 200 mg every 3 weeks. In some embodiments, thepembrolizumab is administered to treat cervical cancer at about 400 mgevery 6 weeks. In some embodiments, the pembrolizumab administration isbegun 1, 2, 3, 4, or 5 days post IL-2 administration. In someembodiments, the pembrolizumab administration is begun 1, 2, or 3 dayspost IL-2 administration. In some embodiments, the pembrolizumab canalso be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., beforeobtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In some embodiments, the pembrolizumab is administered to treathepatocellular carcinoma (HCC) at about 200 mg every 3 weeks. In someembodiments, the pembrolizumab is administered to treat HCC at about 400mg every 6 weeks. In some embodiments, the pembrolizumab administrationis begun 1, 2, 3, 4, or 5 days post IL-2 administration. In someembodiments, the pembrolizumab administration is begun 1, 2, or 3 dayspost IL-2 administration. In some embodiments, the pembrolizumab canalso be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., beforeobtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In some embodiments, the pembrolizumab is administered to treat Merkelcell carcinoma (MCC) at about 200 mg every 3 weeks for adults. In someembodiments, the pembrolizumab is administered to treat MCC at about 400mg every 6 weeks for adults. In some embodiments, the pembrolizumab isadministered to treat MCC at about 2 mg/kg (up to 200 mg) every 3 weeksfor pediatrics. In some embodiments, the pembrolizumab administration isbegun 1, 2, 3, 4, or 5 days post IL-2 administration. In someembodiments, the pembrolizumab administration is begun 1, 2, or 3 dayspost IL-2 administration. In some embodiments, the pembrolizumab canalso be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., beforeobtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient). In some embodiments, the pembrolizumab administration is begun1, 2, 3, 4, or 5 days post IL-2 administration. In some embodiments, thepembrolizumab administration is begun 1, 2, or 3 days post IL-2administration. In some embodiments, the pembrolizumab can also beadministered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., before obtaininga tumor sample from the subject or patient). In some embodiments, thepembrolizumab can also be administered 1, 2, or 3 weeks pre-resection(i.e., before obtaining a tumor sample from the subject or patient).

In some embodiments, the pembrolizumab is administered to treat renalcell carcinoma (RCC) at about 200 mg every 3 weeks. In some embodiments,the pembrolizumab is administered to treat RCC at about 400 mg every 6weeks with axitinib 5 mg orally twice daily. In some embodiments, thepembrolizumab administration is begun 1, 2, 3, 4, or 5 days post IL-2administration. In some embodiments, the pembrolizumab administration isbegun 1, 2, or 3 days post IL-2 administration. In some embodiments, thepembrolizumab can also be administered 1, 2, 3, 4 or 5 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient). In some embodiments, the pembrolizumab can also beadministered 1, 2, or 3 weeks pre-resection (i.e., before obtaining atumor sample from the subject or patient).

In some embodiments, the pembrolizumab is administered to treatendometrial carcinoma at about 200 mg every 3 weeks. In someembodiments, the pembrolizumab is administered to treat endometrialcarcinoma at about 400 mg every 6 weeks with lenvatinib 20 mg orallyonce daily for tumors that are not MSI-H or dMMR. In some embodiments,the pembrolizumab administration is begun 1, 2, 3, 4, or 5 days postIL-2 administration. In some embodiments, the pembrolizumabadministration is begun 1, 2, or 3 days post IL-2 administration. Insome embodiments, the pembrolizumab can also be administered 1, 2, 3, 4or 5 weeks pre-resection (i.e., before obtaining a tumor sample from thesubject or patient). In some embodiments, the pembrolizumab can also beadministered 1, 2, or 3 weeks pre-resection (i.e., before obtaining atumor sample from the subject or patient).

In some embodiments, the pembrolizumab is administered to treat tumormutational burden-high (TMB-H) Cancer at about 200 mg every 3 weeks foradults. In some embodiments, the pembrolizumab is administered to treatTMB-H Cancer at about 400 mg every 6 weeks for adults. In someembodiments, the pembrolizumab is administered to treat TMB-H Cancer atabout 2 mg/kg (up to 200 mg) every 3 weeks for pediatrics. In someembodiments, the pembrolizumab administration is begun 1, 2, 3, 4, or 5days post IL-2 administration. In some embodiments, the pembrolizumabadministration is begun 1, 2, or 3 days post IL-2 administration. Insome embodiments, the pembrolizumab can also be administered 1, 2, 3, 4or 5 weeks pre-resection (i.e., before obtaining a tumor sample from thesubject or patient). In some embodiments, the pembrolizumab can also beadministered 1, 2, or 3 weeks pre-resection (i.e., before obtaining atumor sample from the subject or patient).

In some embodiments, the pembrolizumab is administered to treatcutaneous squamous cell carcinoma (cSCC) at about 200 mg every 3 weeks.In some embodiments, the pembrolizumab is administered to treat cSCC atabout 400 mg every 6 weeks. In some embodiments, the pembrolizumabadministration is begun 1, 2, 3, 4, or 5 days post IL-2 administration.In some embodiments, the pembrolizumab administration is begun 1, 2, or3 days post IL-2 administration. In some embodiments, the pembrolizumabcan also be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In some embodiments, the pembrolizumab is administered to treattriple-negative breast cancer (TNBC) at about 200 mg every 3 weeks. Insome embodiments, the pembrolizumab is administered to treat TNBC atabout 400 mg every 6 weeks. In some embodiments, the pembrolizumabadministration is begun 1, 2, 3, 4, or 5 days post IL-2 administration.In some embodiments, the pembrolizumab administration is begun 1, 2, or3 days post IL-2 administration. In some embodiments, the pembrolizumabcan also be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In an embodiment, if the patient or subject is an adult, i.e., treatmentof adult indications, and additional dosing regimen of 400 mg every 6weeks can be employed. In some embodiments, the pembrolizumabadministration is begun 1, 2, 3, 4, or 5 days post IL-2 administration.In some embodiments, the pembrolizumab administration is begun 1, 2, or3 days post IL-2 administration. In some embodiments, the pembrolizumabcan also be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e.,before obtaining a tumor sample from the subject or patient). In someembodiments, the pembrolizumab can also be administered 1, 2, or 3 weekspre-resection (i.e., before obtaining a tumor sample from the subject orpatient).

In an embodiment, the PD-1 inhibitor is a commercially-availableanti-PD-1 monoclonal antibody, such as anti-m-PD-1 clones J43 (Cat#BE0033-2) and RMP1-14 (Cat #BE0146) (Bio X Cell, Inc., West Lebanon,N.H., USA). A number of commercially-available anti-PD-1 antibodies areknown to one of ordinary skill in the art.

In an embodiment, the PD-1 inhibitor is an antibody disclosed in U.S.Pat. No. 8,354,509 or U.S. Patent Application Publication Nos.2010/0266617 A1, 2013/0108651 A1, 2013/0109843 A2, the disclosures ofwhich are incorporated by reference herein. In an embodiment, the PD-1inhibitor is an anti-PD-1 antibody described in U.S. Pat. Nos.8,287,856, 8,580,247, and 8,168,757 and U.S. Patent ApplicationPublication Nos. 2009/0028857 A1, 2010/0285013 A1, 2013/0022600 A1, and2011/0008369 A1, the teachings of which are hereby incorporated byreference. In another embodiment, the PD-1 inhibitor is an anti-PD-1antibody disclosed in U.S. Pat. No. 8,735,553 B1, the disclosure ofwhich is incorporated herein by reference. In an embodiment, the PD-1inhibitor is pidilizumab, also known as CT-011, which is described inU.S. Pat. No. 8,686,119, the disclosure of which is incorporated byreference herein.

In an embodiment, the PD-1 inhibitor may be a small molecule or apeptide, or a peptide derivative, such as those described in U.S. Pat.Nos. 8,907,053; 9,096,642; and 9,044,442 and U.S. Patent ApplicationPublication No. US 2015/0087581; 1,2,4-oxadiazole compounds andderivatives such as those described in U.S. Patent ApplicationPublication No. 2015/0073024; cyclic peptidomimetic compounds andderivatives such as those described in U.S. Patent ApplicationPublication No. US 2015/0073042; cyclic compounds and derivatives suchas those described in U.S. Patent Application Publication No. US2015/0125491; 1,3,4-oxadiazole and 1,3,4-thiadiazole compounds andderivatives such as those described in International Patent ApplicationPublication No. WO 2015/033301; peptide-based compounds and derivativessuch as those described in International Patent Application PublicationNos. WO 2015/036927 and WO 2015/04490, or a macrocyclic peptide-basedcompounds and derivatives such as those described in U.S. PatentApplication Publication No. US 2014/0294898; the disclosures of each ofwhich are hereby incorporated by reference in their entireties. In anembodiment, the PD-1 inhibitor is cemiplimab, which is commerciallyavailable from Regeneron, Inc.

In an embodiment, the PD-L1 or PD-L2 inhibitor may be any PD-L1 or PD-L2inhibitor, antagonist, or blocker known in the art. In particular, it isone of the PD-L1 or PD-L2 inhibitors, antagonist, or blockers describedin more detail in the following paragraphs. The terms “inhibitor,”“antagonist,” and “blocker” are used interchangeably herein in referenceto PD-L1 and PD-L2 inhibitors. For avoidance of doubt, references hereinto a PD-L1 or PD-L2 inhibitor that is an antibody may refer to acompound or antigen-binding fragments, variants, conjugates, orbiosimilars thereof. For avoidance of doubt, references herein to aPD-L1 or PD-L2 inhibitor may refer to a compound or a pharmaceuticallyacceptable salt, ester, solvate, hydrate, cocrystal, or prodrug thereof.

In some embodiments, the compositions, processes and methods describedherein include a PD-L1 or PD-L2 inhibitor. In some embodiments, thePD-L1 or PD-L2 inhibitor is a small molecule. In a preferred embodiment,the PD-L1 or PD-L2 inhibitor is an antibody (i.e., an anti-PD-1antibody), a fragment thereof, including Fab fragments, or asingle-chain variable fragment (scFv) thereof. In some embodiments thePD-L1 or PD-L2 inhibitor is a polyclonal antibody. In a preferredembodiment, the PD-L1 or PD-L2 inhibitor is a monoclonal antibody. Insome embodiments, the PD-L1 or PD-L2 inhibitor competes for binding withPD-L1 or PD-L2, and/or binds to an epitope on PD-L1 or PD-L2. In anembodiment, the antibody competes for binding with PD-L1 or PD-L2,and/or binds to an epitope on PD-L1 or PD-L2.

In some embodiments, the PD-L1 inhibitors provided herein are selectivefor PD-L1, in that the compounds bind or interact with PD-L1 atsubstantially lower concentrations than they bind or interact with otherreceptors, including the PD-L2 receptor. In certain embodiments, thecompounds bind to the PD-L1 receptor at a binding constant that is atleast about a 2-fold higher concentration, about a 3-fold higherconcentration, about a 5-fold higher concentration, about a 10-foldhigher concentration, about a 20-fold higher concentration, about a30-fold higher concentration, about a 50-fold higher concentration,about a 100-fold higher concentration, about a 200-fold higherconcentration, about a 300-fold higher concentration, or about a500-fold higher concentration than to the PD-L2 receptor.

In some embodiments, the PD-L2 inhibitors provided herein are selectivefor PD-L2, in that the compounds bind or interact with PD-L2 atsubstantially lower concentrations than they bind or interact with otherreceptors, including the PD-L1 receptor. In certain embodiments, thecompounds bind to the PD-L2 receptor at a binding constant that is atleast about a 2-fold higher concentration, about a 3-fold higherconcentration, about a 5-fold higher concentration, about a 10-foldhigher concentration, about a 20-fold higher concentration, about a30-fold higher concentration, about a 50-fold higher concentration,about a 100-fold higher concentration, about a 200-fold higherconcentration, about a 300-fold higher concentration, or about a500-fold higher concentration than to the PD-L1 receptor.

Without being bound by any theory, it is believed that tumor cellsexpress PD-L1, and that T cells express PD-1. However, PD-L1 expressionby tumor cells is not required for efficacy of PD-1 or PD-L1 inhibitorsor blockers. In an embodiment, the tumor cells express PD-L1. In anotherembodiment, the tumor cells do not express PD-L1. In some embodiments,the methods can include a combination of a PD-1 and a PD-L1 antibody,such as those described herein, in combination with a TIL. Theadministration of a combination of a PD-1 and a PD-L1 antibody and a TILmay be simultaneous or sequential.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is one that bindshuman PD-L1 and/or PD-L2 with a KD of about 100 pM or lower, binds humanPD-L1 and/or PD-L2 with a KD of about 90 pM or lower, binds human PD-L1and/or PD-L2 with a KD of about 80 pM or lower, binds human PD-L1 and/orPD-L2 with a KD of about 70 pM or lower, binds human PD-L1 and/or PD-L2with a KD of about 60 pM or lower, a KD of about 50 pM or lower, bindshuman PD-L1 and/or PD-L2 with a KD of about 40 pM or lower, or bindshuman PD-L1 and/or PD-L2 with a KD of about 30 pM or lower,

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is one that bindsto human PD-L1 and/or PD-L2 with a kassoc of about 7.5×105 l/M·s orfaster, binds to human PD-L1 and/or PD-L2 with a kassoc of about 8×105l/M·s or faster, binds to human PD-L1 and/or PD-L2 with a kassoc ofabout 8.5×105 l/M·s or faster, binds to human PD-L1 and/or PD-L2 with akassoc of about 9×105 l/M·s or faster, binds to human PD-L1 and/or PD-L2with a kassoc of about 9.5×105 l/M·s and/or faster, or binds to humanPD-L1 and/or PD-L2 with a kassoc of about 1×106 l/M·s or faster.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is one that bindsto human PD-L1 or PD-L2 with a kdissoc of about 2×10-5 l/s or slower,binds to human PD-1 with a kdissoc of about 2.1×10-5 l/s or slower,binds to human PD-1 with a kdissoc of about 2.2×10-5 l/s or slower,binds to human PD-1 with a kdissoc of about 2.3×10-5 l/s or slower,binds to human PD-1 with a kdissoc of about 2.4×10-5 l/s or slower,binds to human PD-1 with a kdissoc of about 2.5×10-5 l/s or slower,binds to human PD-1 with a kdissoc of about 2.6×10-5 l/s or slower,binds to human PD-L1 or PD-L2 with a kdissoc of about 2.7×10-5 l/s orslower, or binds to human PD-L1 or PD-L2 with a kdissoc of about 3×10-5l/s or slower.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is one that blocksor inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with anIC50 of about 10 nM or lower; blocks or inhibits binding of human PD-L1or human PD-L2 to human PD-1 with an IC50 of about 9 nM or lower; blocksor inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with anIC50 of about 8 nM or lower; blocks or inhibits binding of human PD-L1or human PD-L2 to human PD-1 with an IC50 of about 7 nM or lower; blocksor inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with anIC50 of about 6 nM or lower; blocks or inhibits binding of human PD-L1or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower; blocksor inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with anIC50 of about 4 nM or lower; blocks or inhibits binding of human PD-L1or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower; blocksor inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with anIC50 of about 2 nM or lower; or blocks human PD-1, or blocks binding ofhuman PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 1 nM orlower.

In an embodiment, the PD-L1 inhibitor is durvalumab, also known asMEDI4736 (which is commercially available from Medimmune, LLC,Gaithersburg, Md., a subsidiary of AstraZeneca plc.), or antigen-bindingfragments, conjugates, or variants thereof. In an embodiment, the PD-L1inhibitor is an antibody disclosed in U.S. Pat. No. 8,779,108 or U.S.Patent Application Publication No. 2013/0034559, the disclosures ofwhich are incorporated by reference herein. The clinical efficacy ofdurvalumab has been described in Page, et al., Ann. Rev. Med., 2014, 65,185-202; Brahmer, et al., J. Clin. Oncol. 2014, 32, 5s (supplement,abstract 8021); and McDermott, et al., Cancer Treatment Rev., 2014, 40,1056-64. The preparation and properties of durvalumab are described inU.S. Pat. No. 8,779,108, the disclosure of which is incorporated byreference herein. The amino acid sequences of durvalumab are set forthin Table 20. The durvalumab monoclonal antibody includes disulfidelinkages at 22-96, 22″-96″, 23′-89′, 23′″-89′″, 135′-195′, 135′″-195′″,148-204, 148″-204″, 215′-224, 215′″-224″, 230-230″, 233-233″, 265-325,265″-325″, 371-429, and 371″-429′; and N-glycosylation sites at Asn-301and Asn-301″.

In an embodiment, a PD-L1 inhibitor comprises a heavy chain given by SEQID NO:178 and a light chain given by SEQ ID NO:179. In an embodiment, aPD-L1 inhibitor comprises heavy and light chains having the sequencesshown in SEQ ID NO:178 and SEQ ID NO:179, respectively, or antigenbinding fragments, Fab fragments, single-chain variable fragments(scFv), variants, or conjugates thereof. In an embodiment, a PD-L1inhibitor comprises heavy and light chains that are each at least 99%identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179,respectively. In an embodiment, a PD-L1 inhibitor comprises heavy andlight chains that are each at least 98% identical to the sequences shownin SEQ ID NO:178 and SEQ ID NO:179, respectively. In an embodiment, aPD-L1 inhibitor comprises heavy and light chains that are each at least97% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179,respectively. In an embodiment, a PD-L1 inhibitor comprises heavy andlight chains that are each at least 96% identical to the sequences shownin SEQ ID NO:178 and SEQ ID NO:179, respectively. In an embodiment, aPD-L1 inhibitor comprises heavy and light chains that are each at least95% identical to the sequences shown in SEQ ID NO:178 and SEQ ID NO:179,respectively.

In an embodiment, the PD-L1 inhibitor comprises the heavy and lightchain CDRs or variable regions (VRs) of durvalumab. In an embodiment,the PD-L1 inhibitor heavy chain variable region (V_(H)) comprises thesequence shown in SEQ ID NO:180, and the PD-L1 inhibitor light chainvariable region (V_(L)) comprises the sequence shown in SEQ ID NO:181,or conservative amino acid substitutions thereof. In an embodiment, aPD-L1 inhibitor comprises V_(H) and V_(L) regions that are each at least99% identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO:181,respectively. In an embodiment, a PD-L1 inhibitor comprises V_(H) andV_(L) regions that are each at least 98% identical to the sequencesshown in SEQ ID NO:180 and SEQ ID NO:181, respectively. In anembodiment, a PD-L1 inhibitor comprises V_(H) and V_(L) regions that areeach at least 97% identical to the sequences shown in SEQ ID NO:180 andSEQ ID NO:181, respectively. In an embodiment, a PD-L1 inhibitorcomprises V_(H) and V_(L) regions that are each at least 96% identicalto the sequences shown in SEQ ID NO:180 and SEQ ID NO:181, respectively.In an embodiment, a PD-L1 inhibitor comprises V_(H) and V_(L) regionsthat are each at least 95% identical to the sequences shown in SEQ IDNO:180 and SEQ ID NO:181, respectively.

In an embodiment, a PD-L1 inhibitor comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:182, SEQ IDNO:183, and SEQ ID NO:184, respectively, or conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:185, SEQ ID NO:186, and SEQID NO:187, respectively, or conservative amino acid substitutionsthereof. In an embodiment, the antibody competes for binding with,and/or binds to the same epitope on PD-L1 as any of the aforementionedantibodies.

In an embodiment, the PD-L1 inhibitor is an anti-PD-L1 biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to durvalumab. In an embodiment, the biosimilar comprises ananti-PD-L1 antibody comprising an amino acid sequence which has at least97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, tothe amino acid sequence of a reference medicinal product or referencebiological product and which comprises one or more post-translationalmodifications as compared to the reference medicinal product orreference biological product, wherein the reference medicinal product orreference biological product is durvalumab. In some embodiments, the oneor more post-translational modifications are selected from one or moreof: glycosylation, oxidation, deamidation, and truncation. In someembodiments, the biosimilar is an anti-PD-L1 antibody authorized orsubmitted for authorization, wherein the anti-PD-L1 antibody is providedin a formulation which differs from the formulations of a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is durvalumab. Theanti-PD-L1 antibody may be authorized by a drug regulatory authoritysuch as the U.S. FDA and/or the European Union's EMA. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is durvalumab. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is durvalumab.

TABLE 20Amino acid sequences for PD-L1 inhibitors related to durvalumab.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 178EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYWMSWVRQA PGKGLEWVAN IKQDGSEKYY  60durvalumabVDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREG GWFGELAFDY WGQGTLVTVS 120heavy chainSASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS 180SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPEFEG 240GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPASIEKTI SKAKGQPREP QVYTLPPSRE 360EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR 420WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K 451 SEQ ID NO: 179EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYWMSWVRQA PGKGLEWVAN EIVLTQSPGT  60durvalumabLSLSPGERAT LSCRASQRVS SSYLAWYQQK PGQAPRLLIY DASSRATGIP DRFSGSGSGT 120light chainDFTLTISRLE PEDFAVYYCQ QYGSLPWTFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT 180ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH 240KVYACEVTHQ GLSSPVTKSF NRGEC 265 SEQ ID NO: 180EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYWMSWVRQA PGKGLEWVAN IKQDGSEKYY  60durvalumabVDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREG GWFGELAFDY WGQGTLVTVS 120variable S 121 heavy chain SEQ ID NO: 181EIVLTQSPGT LSLSPGERAT LSCRASQRVS SSYLAWYQQK PGQAPRLLIY DASSRATGIP  60durvalumab DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSLPWTFG QGTKVEIK 108variable light chain SEQ ID NO: 182 RYWMS   5 durvalumab heavy chainCDR1 SEQ ID NO: 183 NIKQDGSEKY YVDSVKG  17 durvalumab heavy chain CDR2SEQ ID NO: 184 EGGWFGELAF DY  12 durvalumab heavy chain CDR3SEQ ID NO: 185 RASQRVSSSY LA  12 durvalumab light chain CDR1SEQ ID NO: 186 DASSRAT   7 durvalumab light chain CDR2 SEQ ID NO: 187QQYGSLPWT   9 durvalumab light chain CDR3

In an embodiment, the PD-L1 inhibitor is avelumab, also known asMSB0010718C (commercially available from Merck KGaA/EMD Serono), orantigen-binding fragments, conjugates, or variants thereof. Thepreparation and properties of avelumab are described in U.S. PatentApplication Publication No. US 2014/0341917 A1, the disclosure of whichis specifically incorporated by reference herein. The amino acidsequences of avelumab are set forth in Table 21. Avelumab hasintra-heavy chain disulfide linkages (C23-C104) at 22-96, 147-203,264-324, 370-428, 22″-96″, 147″-203″, 264″-324″, and 370″-428″;intra-light chain disulfide linkages (C23-C104) at 22′-90′, 138′-197′,22′″-90′″, and 138′″-197′″; intra-heavy-light chain disulfide linkages(h 5-CL 126) at 223-215′ and 223″-215′″; intra-heavy-heavy chaindisulfide linkages (h 11, h 14) at 229-229″ and 232-232″;N-glycosylation sites (H CH2 N84.4) at 300, 300″; fucosylated complexbi-antennary CHO-type glycans; and H CHS K2 C-terminal lysine clippingat 450 and 450′.

In an embodiment, a PD-L1 inhibitor comprises a heavy chain given by SEQID NO:188 and a light chain given by SEQ ID NO:189. In an embodiment, aPD-L1 inhibitor comprises heavy and light chains having the sequencesshown in SEQ ID NO:188 and SEQ ID NO:189, respectively, or antigenbinding fragments, Fab fragments, single-chain variable fragments(scFv), variants, or conjugates thereof. In an embodiment, a PD-L1inhibitor comprises heavy and light chains that are each at least 99%identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189,respectively. In an embodiment, a PD-L1 inhibitor comprises heavy andlight chains that are each at least 98% identical to the sequences shownin SEQ ID NO:188 and SEQ ID NO:189, respectively. In an embodiment, aPD-L1 inhibitor comprises heavy and light chains that are each at least97% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189,respectively. In an embodiment, a PD-L1 inhibitor comprises heavy andlight chains that are each at least 96% identical to the sequences shownin SEQ ID NO:188 and SEQ ID NO:189, respectively. In an embodiment, aPD-L1 inhibitor comprises heavy and light chains that are each at least95% identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO:189,respectively.

In an embodiment, the PD-L1 inhibitor comprises the heavy and lightchain CDRs or variable regions (VRs) of avelumab. In an embodiment, thePD-L1 inhibitor heavy chain variable region (VH) comprises the sequenceshown in SEQ ID NO:190, and the PD-L1 inhibitor light chain variableregion (VL) comprises the sequence shown in SEQ ID NO:191, orconservative amino acid substitutions thereof. In an embodiment, a PD-L1inhibitor comprises V_(H) and V_(L) regions that are each at least 99%identical to the sequences shown in SEQ ID NO:190 and SEQ ID NO:191,respectively. In an embodiment, a PD-L1 inhibitor comprises V_(H) andV_(L) regions that are each at least 98% identical to the sequencesshown in SEQ ID NO:190 and SEQ ID NO:191, respectively. In anembodiment, a PD-L1 inhibitor comprises V_(H) and V_(L) regions that areeach at least 97% identical to the sequences shown in SEQ ID NO:190 andSEQ ID NO:191, respectively. In an embodiment, a PD-L1 inhibitorcomprises V_(H) and V_(L) regions that are each at least 96% identicalto the sequences shown in SEQ ID NO:190 and SEQ ID NO:191, respectively.In an embodiment, a PD-L1 inhibitor comprises V_(H) and V_(L) regionsthat are each at least 95% identical to the sequences shown in SEQ IDNO:190 and SEQ ID NO:191, respectively.

In an embodiment, a PD-L1 inhibitor comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:192, SEQ IDNO:193, and SEQ ID NO:194, respectively, or conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:195, SEQ ID NO:196, and SEQID NO:197, respectively, or conservative amino acid substitutionsthereof. In an embodiment, the antibody competes for binding with,and/or binds to the same epitope on PD-L1 as any of the aforementionedantibodies.

In an embodiment, the PD-L1 inhibitor is an anti-PD-L1 biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to avelumab. In an embodiment, the biosimilar comprises ananti-PD-L1 antibody comprising an amino acid sequence which has at least97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, tothe amino acid sequence of a reference medicinal product or referencebiological product and which comprises one or more post-translationalmodifications as compared to the reference medicinal product orreference biological product, wherein the reference medicinal product orreference biological product is avelumab. In some embodiments, the oneor more post-translational modifications are selected from one or moreof: glycosylation, oxidation, deamidation, and truncation. In someembodiments, the biosimilar is an anti-PD-L1 antibody authorized orsubmitted for authorization, wherein the anti-PD-L1 antibody is providedin a formulation which differs from the formulations of a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is avelumab. Theanti-PD-L1 antibody may be authorized by a drug regulatory authoritysuch as the U.S. FDA and/or the European Union's EMA. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is avelumab. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is avelumab.

TABLE 21 Amino acid sequences for PD-L1 inhibitors related to avelumab.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 188EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYIMMWVRQA PGKGLEWVSS IYPSGGITFY  60avelumabADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARIK LGTVTTVDYW GQGTLVTVSS 120heavy chainASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 160GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 240PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 360LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQ ID NO: 189QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMI YDVSNRPSGV  60avelumabSNRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTRV FGTGTKVTVL GQPKANPTVT 120light chainLFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK AGVETTKPSK QSNNKYAASS 180YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS 216 SEQ ID NO: 190EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYIMMWVRQA PGKGLEWVSS IYPSGGITFY  60avelumabADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARIK LGTVTTVDYW GQGTLVTVSS 120variable heavy chain SEQ ID NO: 191QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMI YDVSNRPSGV  60avelumab SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTRV FGTGTKVTVL 110variable light chain SEQ ID NO: 192 SYIMM   5 avelumab heavy chain CDR1SEQ ID NO: 193 SIYPSGGITF YADTVKG  17 avelumab heavy chain CDR2SEQ ID NO: 194 IKLGTVTTVD Y  11 avelumab heavy chain CDR3 SEQ ID NO: 195TGTSSDVGGY NYVS  14 avelumab light chain CDR1 SEQ ID NO: 196 DVSNRPS   7avelumab light chain CDR2 SEQ ID NO: 197 SSYTSSSTRV  10 avelumablight chain CDR3

In an embodiment, the PD-L1 inhibitor is atezolizumab, also known asMPDL3280A or RG7446 (commercially available as TECENTRIQ from Genentech,Inc., a subsidiary of Roche Holding AG, Basel, Switzerland), orantigen-binding fragments, conjugates, or variants thereof. In anembodiment, the PD-L1 inhibitor is an antibody disclosed in U.S. Pat.No. 8,217,149, the disclosure of which is specifically incorporated byreference herein. In an embodiment, the PD-L1 inhibitor is an antibodydisclosed in U.S. Patent Application Publication Nos. 2010/0203056 A1,2013/0045200 A1, 2013/0045201 A1, 2013/0045202 A1, or 2014/0065135 A1,the disclosures of which are specifically incorporated by referenceherein. The preparation and properties of atezolizumab are described inU.S. Pat. No. 8,217,149, the disclosure of which is incorporated byreference herein. The amino acid sequences of atezolizumab are set forthin Table 22. Atezolizumab has intra-heavy chain disulfide linkages(C23-C104) at 22-96, 145-201, 262-322, 368-426, 22″-96″, 145″-201″,262″-322″, and 368″-426″; intra-light chain disulfide linkages(C23-C104) at 23′-88′, 134′-194′, 23′″-88″, and 134′″-194′″;intra-heavy-light chain disulfide linkages (h 5-CL 126) at 221-214′ and221″-214′″; intra-heavy-heavy chain disulfide linkages (h 11, h 14) at227-227″ and 230-230″; and N-glycosylation sites (H CH2 N84.4>A) at 298and 298′.

In an embodiment, a PD-L1 inhibitor comprises a heavy chain given by SEQID NO:198 and a light chain given by SEQ ID NO:199. In an embodiment, aPD-L1 inhibitor comprises heavy and light chains having the sequencesshown in SEQ ID NO:198 and SEQ ID NO:199, respectively, or antigenbinding fragments, Fab fragments, single-chain variable fragments(scFv), variants, or conjugates thereof. In an embodiment, a PD-L1inhibitor comprises heavy and light chains that are each at least 99%identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199,respectively. In an embodiment, a PD-L1 inhibitor comprises heavy andlight chains that are each at least 98% identical to the sequences shownin SEQ ID NO:198 and SEQ ID NO:199, respectively. In an embodiment, aPD-L1 inhibitor comprises heavy and light chains that are each at least97% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199,respectively. In an embodiment, a PD-L1 inhibitor comprises heavy andlight chains that are each at least 96% identical to the sequences shownin SEQ ID NO:198 and SEQ ID NO:199, respectively. In an embodiment, aPD-L1 inhibitor comprises heavy and light chains that are each at least95% identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO:199,respectively.

In an embodiment, the PD-L1 inhibitor comprises the heavy and lightchain CDRs or variable regions (VRs) of atezolizumab. In an embodiment,the PD-L1 inhibitor heavy chain variable region (V_(H)) comprises thesequence shown in SEQ ID NO:200, and the PD-L1 inhibitor light chainvariable region (V_(L)) comprises the sequence shown in SEQ ID NO:201,or conservative amino acid substitutions thereof. In an embodiment, aPD-L1 inhibitor comprises V_(H) and V_(L) regions that are each at least99% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201,respectively. In an embodiment, a PD-L1 inhibitor comprises Vu and V_(L)regions that are each at least 98% identical to the sequences shown inSEQ ID NO:200 and SEQ ID NO:201, respectively. In an embodiment, a PD-L1inhibitor comprises V_(H) and V_(L) regions that are each at least 97%identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201,respectively. In an embodiment, a PD-L1 inhibitor comprises V_(H) andV_(L) regions that are each at least 96% identical to the sequencesshown in SEQ ID NO:200 and SEQ ID NO:201, respectively. In anembodiment, a PD-L1 inhibitor comprises V_(H) and V_(L) regions that areeach at least 95% identical to the sequences shown in SEQ ID NO:200 andSEQ ID NO:201, respectively.

In an embodiment, a PD-L1 inhibitor comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:202, SEQ IDNO:203, and SEQ ID NO:204, respectively, or conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:205, SEQ ID NO:206, and SEQID NO:207, respectively, or conservative amino acid substitutionsthereof. In an embodiment, the antibody competes for binding with,and/or binds to the same epitope on PD-L1 as any of the aforementionedantibodies.

In an embodiment, the anti-PD-L1 antibody is an anti-PD-L1 biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to atezolizumab. In an embodiment, the biosimilar comprises ananti-PD-L1 antibody comprising an amino acid sequence which has at least97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, tothe amino acid sequence of a reference medicinal product or referencebiological product and which comprises one or more post-translationalmodifications as compared to the reference medicinal product orreference biological product, wherein the reference medicinal product orreference biological product is atezolizumab. In some embodiments, theone or more post-translational modifications are selected from one ormore of: glycosylation, oxidation, deamidation, and truncation. In someembodiments, the biosimilar is an anti-PD-L1 antibody authorized orsubmitted for authorization, wherein the anti-PD-L1 antibody is providedin a formulation which differs from the formulations of a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is atezolizumab. Theanti-PD-L1 antibody may be authorized by a drug regulatory authoritysuch as the U.S. FDA and/or the European Union's EMA. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is atezolizumab. Insome embodiments, the biosimilar is provided as a composition whichfurther comprises one or more excipients, wherein the one or moreexcipients are the same or different to the excipients comprised in areference medicinal product or reference biological product, wherein thereference medicinal product or reference biological product isatezolizumab.

TABLE 22Amino acid sequences for PD-L1 inhibitors related to atezolizumab.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 198EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA PGKGLEWVAW ISPYGGSTYY  60atezolizumabADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 120heavy chainTKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 180YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 240VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 300YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 360KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 420GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 SEQ ID NO: 199DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS  60atezolizumabRFSGSGSGTD FTLTISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKRTV AAPSVFIFPP 120light chainSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ ID NO: 200EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA PGKGLEWVAW ISPYGGSTYY  60atezolizumabADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSA 118variable heavy chain SEQ ID NO: 201DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS  60atezolizumab RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKR 108variable light chain SEQ ID NO: 202 GFTFSDSWIH  10 atezolizumabheavy chain CDR1 SEQ ID NO: 203 AWISPYGGST YYADSVKG  18 atezolizumabheavy chain CDR2 SEQ ID NO: 204 RHWPGGFDY   9 atezolizumab heavy chainCDR3 SEQ ID NO: 205 RASQDVSTAV A  11 atezolizumab light chain CDR1SEQ ID NO: 206 SASFLYS   7 atezolizumab light chain CDR2 SEQ ID NO: 207QQYLYHPAT   9 atezolizumab light chain CDR3

In an embodiment, PD-L1 inhibitors include those antibodies described inU.S. Patent Application Publication No. US 2014/0341917 A1, thedisclosure of which is incorporated by reference herein. In anotherembodiment, antibodies that compete with any of these antibodies forbinding to PD-L1 are also included. In an embodiment, the anti-PD-L1antibody is MDX-1105, also known as BMS-935559, which is disclosed inU.S. Pat. No. 7,943,743, the disclosures of which are incorporated byreference herein. In an embodiment, the anti-PD-L1 antibody is selectedfrom the anti-PD-L1 antibodies disclosed in U.S. Pat. No. 7,943,743,which are incorporated by reference herein.

In an embodiment, the PD-L1 inhibitor is a commercially-availablemonoclonal antibody, such as INVIVOMAB anti-m-PD-L1 clone 10F.9G2(Catalog #BE0101, Bio X Cell, Inc., West Lebanon, N.H., USA). In anembodiment, the anti-PD-L1 antibody is a commercially-availablemonoclonal antibody, such as AFFYMETRIX EBIOSCIENCE (MIH1). A number ofcommercially-available anti-PD-L1 antibodies are known to one ofordinary skill in the art.

In an embodiment, the PD-L2 inhibitor is a commercially-availablemonoclonal antibody, such as BIOLEGEND 24F.10C12 Mouse IgG2a, κ isotype(catalog #329602 Biolegend, Inc., San Diego, Calif.), SIGMA anti-PD-L2antibody (catalog #SAB3500395, Sigma-Aldrich Co., St. Louis, Mo.), orother commercially-available anti-PD-L2 antibodies known to one ofordinary skill in the art.

In some embodiments, the present invention includes a method of treatinga patient with a cancer comprising the steps of administering a TILregimen, wherein the TIL regimen includes a TIL product geneticallymodified to express a CCR, further comprising the step of administeringeither a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, thepresent invention includes a composition comprising (i) a TIL productgenetically modified to express a CCR and (ii) either a PD-1 inhibitoror a PD-L1 inhibitor. In some embodiments, the present inventionincludes a kit comprising (i) a TIL product genetically modified toexpress a CCR and (ii) either a PD-1 inhibitor or a PD-L1 inhibitor.

3. Combinations with CTLA-4 Inhibitors

In some embodiments, the TIL therapy provided to patients with cancermay include treatment with therapeutic populations of TILs alone or mayinclude a combination treatment including TILs and one or more CTLA-4inhibitors.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a member of theimmunoglobulin superfamily and is expressed on the surface of helper Tcells. CTLA-4 is a negative regulator of CD28-dependent T cellactivation and acts as a checkpoint for adaptive immune responses.Similar to the T cell costimulatory protein CD28, the CTLA-4 bindingantigen presents CD80 and CD86 on the cells. CTLA-4 delivers asuppressor signal to T cells, while CD28 delivers a stimulus signal.Human antibodies against human CTLA-4 have been described asimmunostimulatory modulators in many disease conditions, such astreating or preventing viral and bacterial infections and for treatingcancer (WO 01/14424 and WO 00/37504). A number of fully human anti-humanCTLA-4 monoclonal antibodies (mAbs) have been studied in clinical trialsfor the treatment of various types of solid tumors, including, but notlimited to, ipilimumab (MDX-010) and tremelimumab (CP-675,206).

In some embodiments, a CTLA-4 inhibitor may be any CTLA-4 inhibitor orCTLA-4 blocker known in the art. In particular, it is one of the CTLA-4inhibitors or blockers described in more detail in the followingparagraphs. The terms “inhibitor,” “antagonist,” and “blocker” are usedinterchangeably herein in reference to CTLA-4 inhibitors. For avoidanceof doubt, references herein to a CTLA-4 inhibitor that is an antibodymay refer to a compound or antigen-binding fragments, variants,conjugates, or biosimilars thereof. For avoidance of doubt, referencesherein to a CTLA-4 inhibitor may also refer to a small molecule compoundor a pharmaceutically acceptable salt, ester, solvate, hydrate,cocrystal, or prodrug thereof.

Suitable CTLA-4 inhibitors for use in the methods of the invention,include, without limitation, anti-CTLA-4 antibodies, human anti-CTLA-4antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4antibodies, MDX-010 (ipilimumab), tremelimumab, anti-CD28 antibodies,anti-CTLA-4 adnectins, anti-CTLA-4 domain antibodies, single chainanti-CTLA-4 fragments, heavy chain anti-CTLA-4 fragments, light chainanti-CTLA-4 fragments, inhibitors of CTLA-4 that agonize theco-stimulatory pathway, the antibodies disclosed in PCT Publication No.WO 2001/014424, the antibodies disclosed in PCT Publication No. WO2004/035607, the antibodies disclosed in U.S. Publication No.2005/0201994, and the antibodies disclosed in granted European PatentNo. EP 1212422 B1, the disclosures of each of which are incorporatedherein by reference. Additional CTLA-4 antibodies are described in U.S.Pat. Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; in PCTPublication Nos. WO 01/14424 and WO 00/37504; and in U.S. PublicationNos. 2002/0039581 and 2002/086014, the disclosures of each of which areincorporated herein by reference. Other anti-CTLA-4 antibodies that canbe used in a method of the present invention include, for example, thosedisclosed in: WO 98/42752; U.S. Pat. Nos. 6,682,736 and 6,207,156;Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998);Camacho et al., J. Clin. Oncology, 22(145): Abstract No. 2505 (2004)(antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998),and U.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281, thedisclosures of each of which are incorporated herein by reference.

Additional CTLA-4 inhibitors include, but are not limited to, thefollowing: any inhibitor that is capable of disrupting the ability ofCD28 antigen to bind to its cognate ligand, to inhibit the ability ofCTLA-4 to bind to its cognate ligand, to augment T cell responses viathe co-stimulatory pathway, to disrupt the ability of B7 to bind to CD28and/or CTLA-4, to disrupt the ability of B7 to activate theco-stimulatory pathway, to disrupt the ability of CD80 to bind to CD28and/or CTLA-4, to disrupt the ability of CD80 to activate theco-stimulatory pathway, to disrupt the ability of CD86 to bind to CD28and/or CTLA-4, to disrupt the ability of CD86 to activate theco-stimulatory pathway, and to disrupt the co-stimulatory pathway, ingeneral from being activated. This necessarily includes small moleculeinhibitors of CD28, CD80, CD86, CTLA-4, among other members of theco-stimulatory pathway; antibodies directed to CD28, CD80, CD86, CTLA-4,among other members of the co-stimulatory pathway; antisense moleculesdirected against CD28, CD80, CD86, CTLA-4, among other members of theco-stimulatory pathway; adnectins directed against CD28, CD80, CD86,CTLA-4, among other members of the co-stimulatory pathway, RNAiinhibitors (both single and double stranded) of CD28, CD80, CD86,CTLA-4, among other members of the co-stimulatory pathway, among otherCTLA-4 inhibitors.

In some embodiments a CTLA-4 inhibitor binds to CTLA-4 with a K_(d) ofabout 10⁻⁶ M or less, 10⁻⁷M or less, 10⁻⁸ M or less, 10⁻⁹M or less,10⁻¹⁰ M or less, 10⁻¹¹M or less, 10⁻¹² M or less, e.g., between 10⁻¹³ Mand 10⁻¹⁶ M, or within any range having any two of the aforementionedvalues as endpoints. In some embodiments a CTLA-4 inhibitor binds toCTLA-4 with a Kd of no more than 10-fold that of ipilimumab, whencompared using the same assay. In some embodiments a CTLA-4 inhibitorbinds to CTLA-4 with a Kd of about the same as, or less (e.g., up to10-fold lower, or up to 100-fold lower) than that of ipilimumab, whencompared using the same assay. In some embodiments, the IC50 values forinhibition by a CTLA-4 inhibitor of CTLA-4 binding to CD80 or CD86 is nomore than 10-fold greater than that of ipilimumab-mediated inhibition ofCTLA-4 binding to CD80 or CD86, respectively, when compared using thesame assay. In some embodiments, the IC50 values for inhibition by aCTLA-4 inhibitor of CTLA-4 binding to CD80 or CD86 is about the same orless (e.g., up to 10-fold lower, or up to 100-fold lower) than that ofipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86,respectively, when compared using the same assay.

In some embodiments a CTLA-4 inhibitor is used in an amount sufficientto inhibit expression and/or decrease biological activity of CTLA-4 byat least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relativeto a suitable control, e.g., between 50% and 75%, 75% and 90%, or 90%and 100%. In some embodiments a CTLA-4 pathway inhibitor is used in anamount sufficient to decrease the biological activity of CTLA-4 byreducing binding of CTLA-4 to CD80, CD86, or both by at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to a suitablecontrol, e.g., between 50% and 75%, 75% and 90%, or 90% and 100%relative to a suitable control. A suitable control in the context ofassessing or quantifying the effect of an agent of interest is typicallya comparable biological system (e.g., cells or a subject) that has notbeen exposed to or treated with the agent of interest, e.g., CTLA-4pathway inhibitor (or has been exposed to or treated with a negligibleamount). In some embodiments a biological system may serve as its owncontrol (e.g., the biological system may be assessed before exposure toor treatment with the agent and compared with the state after exposureor treatment has started or finished. In some embodiments a historicalcontrol may be used.

In an embodiment, the CTLA-4 inhibitor is ipilimumab (commerciallyavailable as Yervoy from Bristol-Myers Squibb Co.), or biosimilars,antigen-binding fragments, conjugates, or variants thereof. As is knownin the art, ipilimumab refers to an anti-CTLA-4 antibody, a fully humanIgG 1κ antibody derived from a transgenic mouse with human genesencoding heavy and light chains to generate a functional humanrepertoire. is there. Ipilimumab can also be referred to by its CASRegistry Number 477202-00-9, and in PCT Publication Number WO 01/14424,which is incorporated herein by reference in its entirety for allpurposes. It is disclosed as antibody 10DI. Specifically, ipilimumabcontains a light chain variable region and a heavy chain variable region(having a light chain variable region comprising SEQ ID NO:211 andhaving a heavy chain variable region comprising SEQ ID NO:210). Apharmaceutical composition of ipilimumab includes all pharmaceuticallyacceptable compositions containing ipilimumab and one or more diluents,vehicles, or excipients. An example of a pharmaceutical compositioncontaining ipilimumab is described in International Patent ApplicationPublication No. WO 2007/67959. Ipilimumab can be administeredintravenously (IV).

In an embodiment, a CTLA-4 inhibitor comprises a heavy chain given bySEQ ID NO:208 and a light chain given by SEQ ID NO:209. In anembodiment, a CTLA-4 inhibitor comprises heavy and light chains havingthe sequences shown in SEQ ID NO:208 and SEQ ID NO:209, respectively, orantigen binding fragments, Fab fragments, single-chain variablefragments (scFv), variants, or conjugates thereof. In an embodiment, aCTLA-4 inhibitor comprises heavy and light chains that are each at least99% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209,respectively. In an embodiment, a CTLA-4 inhibitor comprises heavy andlight chains that are each at least 98% identical to the sequences shownin SEQ ID NO:208 and SEQ ID NO:209, respectively. In an embodiment, aCTLA-4 inhibitor comprises heavy and light chains that are each at least97% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209,respectively. In an embodiment, a CTLA-4 inhibitor comprises heavy andlight chains that are each at least 96% identical to the sequences shownin SEQ ID NO:208 and SEQ ID NO:209, respectively. In an embodiment, aCTLA-4 inhibitor comprises heavy and light chains that are each at least95% identical to the sequences shown in SEQ ID NO:208 and SEQ ID NO:209,respectively.

In an embodiment, the CTLA-4 inhibitor comprises the heavy and lightchain CDRs or variable regions (VRs) of ipilimumab. In an embodiment,the CTLA-4 inhibitor heavy chain variable region (V_(H)) comprises thesequence shown in SEQ ID NO:210, and the CTLA-4 inhibitor light chainvariable region (V_(L)) comprises the sequence shown in SEQ ID NO:211,or conservative amino acid substitutions thereof. In an embodiment, aCTLA-4 inhibitor comprises V_(H) and V_(L) regions that are each atleast 99% identical to the sequences shown in SEQ ID NO:210 and SEQ IDNO:211, respectively. In an embodiment, a CTLA-4 inhibitor comprisesV_(H) and V_(L) regions that are each at least 98% identical to thesequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively. In anembodiment, a CTLA-4 inhibitor comprises V_(H) and V_(L) regions thatare each at least 97% identical to the sequences shown in SEQ ID NO:210and SEQ ID NO:211, respectively. In an embodiment, a CTLA-4 inhibitorcomprises V_(H) and V_(L) regions that are each at least 96% identicalto the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively.In an embodiment, a CTLA-4 inhibitor comprises V_(H) and V_(L) regionsthat are each at least 95% identical to the sequences shown in SEQ IDNO:210 and SEQ ID NO:211, respectively.

In an embodiment, a CTLA-4 inhibitor comprises the heavy chain CDR1,CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:212,SEQ ID NO:213, and SEQ ID NO:214, respectively, or conservative aminoacid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:215, SEQ ID NO:216, and SEQID NO:217, respectively, or conservative amino acid substitutionsthereof. In an embodiment, the antibody competes for binding with,and/or binds to the same epitope on CTLA-4 as any of the aforementionedantibodies.

In an embodiment, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonalantibody approved by drug regulatory authorities with reference toipilimumab. In an embodiment, the biosimilar comprises an anti-CTLA-4antibody comprising an amino acid sequence which has at least 97%sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to theamino acid sequence of a reference medicinal product or referencebiological product and which comprises one or more post-translationalmodifications as compared to the reference medicinal product orreference biological product, wherein the reference medicinal product orreference biological product is ipilimumab. In some embodiments, the oneor more post-translational modifications are selected from one or moreof: glycosylation, oxidation, deamidation, and truncation. The aminoacid sequences of ipilimumab are set forth in Table 23. In someembodiments, the biosimilar is an anti-CTLA-4 antibody authorized orsubmitted for authorization, wherein the anti-CTLA-4 antibody isprovided in a formulation which differs from the formulations of areference medicinal product or reference biological product, wherein thereference medicinal product or reference biological product isipilimumab. The anti-CTLA-4 antibody may be authorized by a drugregulatory authority such as the U.S. FDA and/or the European Union'sEMA. In some embodiments, the biosimilar is provided as a compositionwhich further comprises one or more excipients, wherein the one or moreexcipients are the same or different to the excipients comprised in areference medicinal product or reference biological product, wherein thereference medicinal product or reference biological product isipilimumab. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is ipilimumab.

TABLE 23 Amino acid sequences for ipilimumab. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 208QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYTMHWVRQA PGKGLEWVTF ISYDGNNKYY  60ipilimumabADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAIYYCARTG WLGPFDYWGQ GTLVTVSSAS 120heavy chainTKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 180YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CDKTH 225 SEQ ID NO: 209EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLAWYQQK PGQAPRLLIY GAFSRATGIP  60ipilimumabDRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIKRT VAAPSVFIFP 120light chainPSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL 180TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC 215 SEQ ID NO: 210QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYTMHWVRQA PGKGLEWVTF ISYDGNNKYY  60ipilimumabADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAIYYCARTG WLGPFDYWGQ GTLVTVSS 118variable heavy chain SEQ ID NO: 211EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLAWYQQK PGQAPRLLIY GAFSRATGIP  60ipilimumab DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIK 108variable light chain SEQ ID NO: 212 GFTFSSYT   8 ipilimumab heavy chainCDR1 SEQ ID NO: 213 TFISYDGNNK  10 ipilimumab heavy chain CDR2SEQ ID NO: 214 ARTGWLGPFD Y  11 ipilimumab heavy chain CDR3SEQ ID NO: 215 QSVGSSY   7 ipilimumab light chain CDR1 SEQ ID NO: 216GAF   3 ipilimumab light chain CDR2 SEQ ID NO: 217 QQYGSSPWT   9ipilimumab light chain CDR3

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilarthereof, and the ipilimumab is administered at a dose of about 0.5 mg/kgto about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor isipilimumab or a biosimilar thereof, and the ipilimumab is administeredat a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg,about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In someembodiments, the ipilimumab administration is begun 1, 2, 3, 4, or 5weeks pre-resection (i.e., prior to obtaining the tumor sample from thesubject or patient). In some embodiments, the ipilimumab administrationis begun 1, 2, or 3 weeks pre-resection (i.e., prior to obtaining thetumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilarthereof, and the ipilimumab is administered at a dose of about 200 mg toabout 500 mg. In some embodiments, the CTLA-4 inhibitor is ipilimumab ora biosimilar thereof, and the ipilimumab is administered at a dose ofabout 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg,about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg,about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, orabout 500 mg. In some embodiments, the ipilimumab administration isbegun 1, 2, 3, 4, or 5 weeks pre-resection (i.e., prior to obtaining thetumor sample from the subject or patient). In some embodiments, theipilimumab administration is begun 1, 2, or 3 weeks pre-resection (i.e.,prior to obtaining the tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilarthereof, and the ipilimumab is administered every 2 weeks, every 3weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In someembodiments, the ipilimumab administration is begun 1, 2, 3, 4, or 5weeks pre-resection (i.e., prior to obtaining the tumor sample from thesubject or patient). In some embodiments, the ipilimumab administrationis begun 1, 2, or 3 weeks pre-resection (i.e., prior to obtaining thetumor sample from the subject or patient).

In some embodiments, the ipilimumab is administered to treatunresectable or metastatic melanoma. In some embodiments, the ipilimumabis administered to treat Unresectable or Metastatic Melanoma at aboutmg/kg every 3 weeks for a maximum of 4 doses. In some embodiments, theipilimumab administration is begun 1, 2, 3, 4, or 5 weeks pre-resection(i.e., prior to obtaining the tumor sample from the subject or patient).In some embodiments, the ipilimumab administration is begun 1, 2, or 3weeks pre-resection (i.e., prior to obtaining the tumor sample from thesubject or patient).

In some embodiments, the ipilimumab is administered for the adjuvanttreatment of melanoma. In some embodiments, the ipilimumab isadministered to for the adjuvant treatment of melanoma at about 10 mg/kgevery 3 weeks for 4 doses, followed by 10 mg/kg every 12 weeks for up to3 years. In some embodiments, the ipilimumab administration is begun 1,2, 3, 4, or 5 weeks pre-resection (i.e., prior to obtaining the tumorsample from the subject or patient). In some embodiments, the ipilimumabadministration is begun 1, 2, or 3 weeks pre-resection (i.e., prior toobtaining the tumor sample from the subject or patient).

In some embodiments, the ipilimumab is administered to treat advancedrenal cell carcinoma. In some embodiments, the ipilimumab isadministered to treat advanced renal cell carcinoma at about 1 mg/kgimmediately following nivolumab 3 mg/kg on the same day, every 3 weeksfor 4 doses. In some embodiments, after completing 4 doses of thecombination, nivolumab can be administered as a single agent accordingto standard dosing regimens for advanced renal cell carcinoma and/orrenal cell carcinoma. In some embodiments, the ipilimumab administrationis begun 1, 2, 3, 4, or 5 weeks pre-resection (i.e., prior to obtainingthe tumor sample from the subject or patient). In some embodiments, theipilimumab administration is begun 1, 2, or 3 weeks pre-resection (i.e.,prior to obtaining the tumor sample from the subject or patient).

In some embodiments, the ipilimumab is administered to treatmicrosatellite instability-high (MSI-H) or mismatch repair deficient(dMMR) metastatic colorectal cancer. In some embodiments, the ipilimumabis administered to treat microsatellite instability-high (MSI-H) ormismatch repair deficient (dMMR) metastatic colorectal cancer at about 1mg/kg intravenously over 30 minutes immediately following nivolumab 3mg/kg intravenously over 30 minutes on the same day, every 3 weeks for 4doses. In some embodiments, after completing 4 doses of the combination,administer nivolumab as a single agent as recommended according tostandard dosing regimens for microsatellite instability-high (MSI-H) ormismatch repair deficient (dMMR) metastatic colorectal cancer. In someembodiments, the ipilimumab administration is begun 1, 2, 3, 4, or 5weeks pre-resection (i.e., prior to obtaining the tumor sample from thesubject or patient). In some embodiments, the ipilimumab administrationis begun 1, 2, or 3 weeks pre-resection (i.e., prior to obtaining thetumor sample from the subject or patient).

In some embodiments, the ipilimumab is administered to treathepatocellular carcinoma. In some embodiments, the ipilimumab isadministered to treat hepatocellular carcinoma at about 3 mg/kgintravenously over 30 minutes immediately following nivolumab 1 mg/kgintravenously over 30 minutes on the same day, every 3 weeks for 4doses. In some embodiments, after completion 4 doses of the combination,administer nivolumab as a single agent according to standard dosingregimens for hepatocellular carcinoma. In some embodiments, theipilimumab administration is begun 1, 2, 3, 4, or 5 weeks pre-resection(i.e., prior to obtaining the tumor sample from the subject or patient).In some embodiments, the ipilimumab administration is begun 1, 2, or 3weeks pre-resection (i.e., prior to obtaining the tumor sample from thesubject or patient).

In some embodiments, the ipilimumab is administered to treat metastaticnon-small cell lung cancer. In some embodiments, the ipilimumab isadministered to treat metastatic non-small cell lung cancer at about 1mg/kg every 6 weeks with nivolumab 3 mg/kg every 2 weeks. In someembodiments, the ipilimumab is administered to treat metastaticnon-small cell lung cancer at about 1 mg/kg every 6 weeks with nivolumab360 mg every 3 weeks and 2 cycles of platinum-doublet chemotherapy. Insome embodiments, the ipilimumab administration is begun 1, 2, 3, 4, or5 weeks pre-resection (i.e., prior to obtaining the tumor sample fromthe subject or patient). In some embodiments, the ipilimumabadministration is begun 1, 2, or 3 weeks pre-resection (i.e., prior toobtaining the tumor sample from the subject or patient).

In some embodiments, the ipilimumab is administered to treat malignantpleural mesothelioma. In some embodiments, the ipilimumab isadministered to treat malignant pleural mesothelioma at about 1 mg/kgevery 6 weeks with nivolumab 360 mg every 3 weeks. In some embodiments,the ipilimumab administration is begun 1, 2, 3, 4, or 5 weekspre-resection (i.e., prior to obtaining the tumor sample from thesubject or patient). In some embodiments, the ipilimumab administrationis begun 1, 2, or 3 weeks pre-resection (i.e., prior to obtaining thetumor sample from the subject or patient).

Tremelimumab (also known as CP-675,206) is a fully human IgG2 monoclonalantibody and has the CAS number 745013-59-6. Tremelimumab is disclosedas antibody 11.2.1 in U.S. Pat. No. 6,682,736 (incorporated herein byreference). The amino acid sequences of the heavy chain and light chainof tremelimumab are set forth in SEQ ID NOs:218 and 219, respectively.Tremelimumab has been investigated in clinical trials for the treatmentof various tumors, including melanoma and breast cancer; in whichTremelimumab was administered intravenously either as single dose ormultiple doses every 4 or 12 weeks at the dose range of 0.01 and 15mg/kg. In the regimens provided by the present invention, tremelimumabis administered locally, particularly intradermally or subcutaneously.The effective amount of tremelimumab administered intradermally orsubcutaneously is typically in the range of 5-200 mg/dose per person. Insome embodiments, the effective amount of tremelimumab is in the rangeof 10-150 mg/dose per person per dose. In some particular embodiments,the effective amount of tremelimumab is about 10, 25, 37.5, 40, 50, 75,100, 125, 150, 175, or 200 mg/dose per person.

In an embodiment, a CTLA-4 inhibitor comprises a heavy chain given bySEQ ID NO:218 and a light chain given by SEQ ID NO:219. In anembodiment, a CTLA-4 inhibitor comprises heavy and light chains havingthe sequences shown in SEQ ID NO:218 and SEQ ID NO:219, respectively, orantigen binding fragments, Fab fragments, single-chain variablefragments (scFv), variants, or conjugates thereof. In an embodiment, aCTLA-4 inhibitor comprises heavy and light chains that are each at least99% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219,respectively. In an embodiment, a CTLA-4 inhibitor comprises heavy andlight chains that are each at least 98% identical to the sequences shownin SEQ ID NO:218 and SEQ ID NO:219, respectively. In an embodiment, aCTLA-4 inhibitor comprises heavy and light chains that are each at least97% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219,respectively. In an embodiment, a CTLA-4 inhibitor comprises heavy andlight chains that are each at least 96% identical to the sequences shownin SEQ ID NO:218 and SEQ ID NO:219, respectively. In an embodiment, aCTLA-4 inhibitor comprises heavy and light chains that are each at least95% identical to the sequences shown in SEQ ID NO:218 and SEQ ID NO:219,respectively.

In an embodiment, the CTLA-4 inhibitor comprises the heavy and lightchain CDRs or variable regions (VRs) of tremelimumab. In an embodiment,the CTLA-4 inhibitor heavy chain variable region (V_(H)) comprises thesequence shown in SEQ ID NO:220, and the CTLA-4 inhibitor light chainvariable region (V_(L)) comprises the sequence shown in SEQ ID NO:221,or conservative amino acid substitutions thereof. In an embodiment, aCTLA-4 inhibitor comprises V_(H) and V_(L) regions that are each atleast 99% identical to the sequences shown in SEQ ID NO:220 and SEQ IDNO:221, respectively. In an embodiment, a CTLA-4 inhibitor comprisesV_(H) and V_(L) regions that are each at least 98% identical to thesequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively. In anembodiment, a CTLA-4 inhibitor comprises V_(H) and V_(L) regions thatare each at least 97% identical to the sequences shown in SEQ ID NO:220and SEQ ID NO:221, respectively. In an embodiment, a CTLA-4 inhibitorcomprises V_(H) and V_(L) regions that are each at least 96% identicalto the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively.In an embodiment, a CTLA-4 inhibitor comprises V_(H) and V_(L) regionsthat are each at least 95% identical to the sequences shown in SEQ IDNO:220 and SEQ ID NO:221, respectively.

In an embodiment, a CTLA-4 inhibitor comprises the heavy chain CDR1,CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:222,SEQ ID NO:223, and SEQ ID NO:224, respectively, or conservative aminoacid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:225, SEQ ID NO:226, and SEQID NO:227, respectively, or conservative amino acid substitutionsthereof. In an embodiment, the antibody competes for binding with,and/or binds to the same epitope on CTLA-4 as any of the aforementionedantibodies.

In an embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to tremelimumab. In an embodiment, the biosimilar comprises ananti-CTLA-4 antibody comprising an amino acid sequence which has atleast 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequenceidentity, to the amino acid sequence of a reference medicinal product orreference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is tremelimumab. In someembodiments, the one or more post-translational modifications areselected from one or more of: glycosylation, oxidation, deamidation, andtruncation. The amino acid sequences of tremelimumab are set forth inTable 24. In some embodiments, the biosimilar is an anti-CTLA-4 antibodyauthorized or submitted for authorization, wherein the anti-CTLA-4antibody is provided in a formulation which differs from theformulations of a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is tremelimumab. The anti-CTLA-4 antibody may be authorized by adrug regulatory authority such as the U.S. FDA and/or the EuropeanUnion's EMA. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is tremelimumab. In some embodiments, the biosimilar is providedas a composition which further comprises one or more excipients, whereinthe one or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is tremelimumab.

TABLE 24 Amino acid sequences for tremelimumab. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 218QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYGMHWVRQA PGKGLEWVAV IWYDGSNKYY  60tremelimumabADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDP RGATLYYYYY GMDVWGQGTT 120heavy chainVTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA 180VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE CPPCPAPPVA 240GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVQFN WYVDGVEVHN AKTKPREEQF 300NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK GLPAPIEKTI SKTKGQPREP QVYTLPPSRE 360EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR 420WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K 451 SEQ ID NO: 219DIQMTQSPSS LSASVGDRVT ITCRASQSIN SYLDWYQQKP GKAPKLLIYA ASSLQSGVPS  60tremelimumabRFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYSTPFTFGP GTKVEIKRTV AAPSVFIFPP 120light  chainSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ ID NO: 220GVVQPGRSLR LSCAASGFTF SSYGMHWVRQ APGKGLEWVA VIWYDGSNKY YADSVKGRFT  60tremelimumabISRDNSKNTL YLQMNSLRAE DTAVYYCARD PRGATLYYYY YGMDVWGQGT TVTVSSASTK 120variable heavy GPSVFPLAPC SRSTSESTAA LGCLVKDYFP EPVTVSWNSG ALTSGVH 167chain SEQ ID NO: 221PSSLSASVGD RVTITCRASQ SINSYLDWYQ QKPGKAPKLL IYAASSLQSG VPSRFSGSGS  60tremelimumabGTDFTLTISS LQPEDFATYY CQQYYSTPFT FGPGTKVEIK RTVAAPSVFI FPPSDEQLKS 120variable light GTASVVCLLN NFYPREAKV 139 chain SEQ ID NO: 222 GFTFSSYGMH 10 tremelimumab heavy chain CDR1 SEQ ID NO: 223 VIWYDGSNKY YADSV  15tremelimumab heavy chain CDR2 SEQ ID NO: 224 DPRGATLYYY YYGMDV  16tremelimumab heavy chain CDR3 SEQ ID NO: 225 RASQSINSYL D  11tremelimumab light chain CDR1 SEQ ID NO: 226 AASSLQS   7 tremelimumablight chain CDR2 SEQ ID NO: 227 QQYYSTPFT   9 tremelimumab light chainCDR3

In some embodiments, the CTLA-4 inhibitor is tremelimumab or abiosimilar thereof, and the tremelimumab is administered at a dose ofabout 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4inhibitor is tremelimumab or a biosimilar thereof, and the tremelimumabis administered at a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg,about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg,about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. Insome embodiments, the tremelimumab administration is begun 1, 2, 3, 4,or 5 weeks pre-resection (i.e., prior to obtaining the tumor sample fromthe subject or patient). In some embodiments, the tremelimumabadministration is begun 1, 2, or 3 weeks pre-resection (i.e., prior toobtaining the tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is tremelimumab or abiosimilar thereof, and the tremelimumab is administered at a dose ofabout 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitoris tremelimumab or a biosimilar thereof, and the tremelimumab isadministered at a dose of about 200 mg, about 220 mg, about 240 mg,about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg,about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg,about 460 mg, about 480 mg, or about 500 mg. In some embodiments, thetremelimumab administration is begun 1, 2, 3, 4, or 5 weekspre-resection (i.e., prior to obtaining the tumor sample from thesubject or patient). In some embodiments, the tremelimumabadministration is begun 1, 2, or 3 weeks pre-resection (i.e., prior toobtaining the tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is tremelimumab or abiosimilar thereof, and the tremelimumab is administered every 2 weeks,every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In someembodiments, the tremelimumab administration is begun 1, 2, 3, 4, or 5weeks pre-resection (i.e., prior to obtaining the tumor sample from thesubject or patient). In some embodiments, the tremelimumabadministration is begun 1, 2, or 3 weeks pre-resection (i.e., prior toobtaining the tumor sample from the subject or patient).

In an embodiment, the CTLA-4 inhibitor is zalifrelimab from Agenus, orbiosimilars, antigen-binding fragments, conjugates, or variants thereof.Zalifrelimab is a fully human monoclonal antibody. Zalifrelimab isassigned Chemical Abstracts Service (CAS) registry number 2148321-69-9and is also known as also known as AGEN1884. The preparation andproperties of zalifrelimab are described in U.S. Pat. No. 10,144,779 andUS Patent Application Publication No. US2020/0024350 A1, the disclosuresof which are incorporated by reference herein.

In an embodiment, a CTLA-4 inhibitor comprises a heavy chain given bySEQ ID NO:228 and a light chain given by SEQ ID NO:229. In anembodiment, a CTLA-4 inhibitor comprises heavy and light chains havingthe sequences shown in SEQ ID NO:228 and SEQ ID NO:229, respectively, orantigen binding fragments, Fab fragments, single-chain variablefragments (scFv), variants, or conjugates thereof. In an embodiment, aCTLA-4 inhibitor comprises heavy and light chains that are each at least99% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229,respectively. In an embodiment, a CTLA-4 inhibitor comprises heavy andlight chains that are each at least 98% identical to the sequences shownin SEQ ID NO:228 and SEQ ID NO:229, respectively. In an embodiment, aCTLA-4 inhibitor comprises heavy and light chains that are each at least97% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229,respectively. In an embodiment, a CTLA-4 inhibitor comprises heavy andlight chains that are each at least 96% identical to the sequences shownin SEQ ID NO:228 and SEQ ID NO:229, respectively. In an embodiment, aCTLA-4 inhibitor comprises heavy and light chains that are each at least95% identical to the sequences shown in SEQ ID NO:228 and SEQ ID NO:229,respectively.

In an embodiment, the CTLA-4 inhibitor comprises the heavy and lightchain CDRs or variable regions (VRs) of zalifrelimab. In an embodiment,the CTLA-4 inhibitor heavy chain variable region (V_(H)) comprises thesequence shown in SEQ ID NO:230, and the CTLA-4 inhibitor light chainvariable region (V_(L)) comprises the sequence shown in SEQ ID NO:231,or conservative amino acid substitutions thereof. In an embodiment, aCTLA-4 inhibitor comprises V_(H) and V_(L) regions that are each atleast 99% identical to the sequences shown in SEQ ID NO:230 and SEQ IDNO:231, respectively. In an embodiment, a CTLA-4 inhibitor comprisesV_(H) and V_(L) regions that are each at least 98% identical to thesequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively. In anembodiment, a CTLA-4 inhibitor comprises V_(H) and V_(L) regions thatare each at least 97% identical to the sequences shown in SEQ ID NO:230and SEQ ID NO:231, respectively. In an embodiment, a CTLA-4 inhibitorcomprises V_(H) and V_(L) regions that are each at least 96% identicalto the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively.In an embodiment, a CTLA-4 inhibitor comprises V_(H) and V_(L) regionsthat are each at least 95% identical to the sequences shown in SEQ IDNO:230 and SEQ ID NO:231, respectively.

In an embodiment, a CTLA-4 inhibitor comprises the heavy chain CDR1,CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:231,SEQ ID NO:233, and SEQ ID NO:234, respectively, or conservative aminoacid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:235, SEQ ID NO:236, and SEQID NO:237, respectively, or conservative amino acid substitutionsthereof. In an embodiment, the antibody competes for binding with,and/or binds to the same epitope on CTLA-4 as any of the aforementionedantibodies.

In an embodiment, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonalantibody approved by drug regulatory authorities with reference tozalifrelimab. In an embodiment, the biosimilar comprises an anti-CTLA-4antibody comprising an amino acid sequence which has at least 97%sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to theamino acid sequence of a reference medicinal product or referencebiological product and which comprises one or more post-translationalmodifications as compared to the reference medicinal product orreference biological product, wherein the reference medicinal product orreference biological product is zalifrelimab. In some embodiments, theone or more post-translational modifications are selected from one ormore of: glycosylation, oxidation, deamidation, and truncation. Theamino acid sequences of zalifrelimab are set forth in Table 25. In someembodiments, the biosimilar is an anti-CTLA-4 antibody authorized orsubmitted for authorization, wherein the anti-CTLA-4 antibody isprovided in a formulation which differs from the formulations of areference medicinal product or reference biological product, wherein thereference medicinal product or reference biological product iszalifrelimab. The anti-CTLA-4 antibody may be authorized by a drugregulatory authority such as the U.S. FDA and/or the European Union'sEMA. In some embodiments, the biosimilar is provided as a compositionwhich further comprises one or more excipients, wherein the one or moreexcipients are the same or different to the excipients comprised in areference medicinal product or reference biological product, wherein thereference medicinal product or reference biological product iszalifrelimab. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is zalifrelimab.

TABLE 25 Amino acid sequences for zalifrelimab. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 228EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYY  60zalifrelimabADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVG LMGPFDIWGQ GTMVTVSSAS 120heavy chainTKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 160YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPELLGGPS 240VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 360KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 420GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 SEQ ID NO: 229EIVLTQSPGT LSLSPGERAT LSCRASQSVS RYLGWYQQKP GQAPRLLIYG ASTRATGIPD  60zalifrelimabRFSGSGSGTD FTLTITRLEP EDFAVYYCQQ YGSSPWTFGQ GTKVEIKRTV AAPSVFIFPP 120light chainSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ ID NO: 230EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYY  60zalifrelimabADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVG LMGPFDIWGQ GTMVTVSS 118variable heavy chain SEQ ID NO: 231EIVLTQSPGT LSLSPGERAT LSCRASQSVS RYLGWYQQKP GQAPRLLIYG ASTRATGIPD  60zalifrelimab RFSGSGSGTD FTLTITRLEP EDFAVYYCQQ YGSSPWTFGQ GTKVEIK 107variable light chain SEQ ID NO: 232 GFTFSSYS   8 zalifrelimabheavy chain CDR1 SEQ ID NO: 233 ISSSSSYI   8 zalifrelimab heavy chainCDR2 SEQ ID NO: 234 ARVGLMGPFD I  11 zalifrelimab heavy chain CDR3SEQ ID NO: 235 QSVSRY   6 zalifrelimab light chain CDR1 SEQ ID NO: 236GAS   3 zalifrelimab light chain CDR2 SEQ ID NO: 237 QQYGSSPWT   9zalifrelimab light chain CDR3

Examples of additional anti-CTLA-4 antibodies includes, but are notlimited to: AGEN1181, BMS-986218, BCD-145, ONC-392, CS1002, REGN4659,and ADG116, which are known to one of ordinary skill in the art.

In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibodydisclosed in any of the following patent publications: US 2019/0048096A1; US 2020/0223907; US 2019/0201334; US 2019/0201334; US 2005/0201994;EP 1212422 B1; WO 2018/204760; WO 2018/204760; WO 2001/014424; WO2004/035607; WO 2003/086459; WO 2012/120125; WO 2000/037504; WO2009/100140; WO 2006/09649; WO2005092380; WO 2007/123737; WO2006/029219; WO 2010/0979597; WO 2006/12168; and WO1997020574, each ofwhich is incorporated herein by reference. Additional CTLA-4 antibodiesare described in U.S. Pat. Nos. 5,811,097, 5,855,887, 6,051,227, and6,984,720; in PCT Publication Nos. WO 01/14424 and WO 00/37504; and inU.S. Publication Nos. 2002/0039581 and 2002/086014; and/or U.S. Pat.Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281, each of which isincorporated herein by reference. In some embodiments, the anti-CTLA-4antibody is, for example, those disclosed in: WO 98/42752; U.S. Pat.Nos. 6,682,736 and 6,207,156; Hurwitz, et al., Proc. Natl. Acad. Sci.USA, 1998, 95, 10067-10071 (1998); Camacho, et al., J. Clin. Oncol.,2004, 22, 145 (Abstract No. 2505 (2004) (antibody CP-675206); or Mokyr,et al., Cancer Res., 1998, 58, 5301-5304 (1998), each of which isincorporated herein by reference.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 ligand asdisclosed in WO 1996/040915 (incorporated herein by reference).

In some embodiments, the CTLA-4 inhibitor is a nucleic acid inhibitor ofCTLA-4 expression. For example, anti-CTLA-4 RNAi molecules may take theform of the molecules described in PCT Publication Nos. WO 1999/032619and WO 2001/029058; U.S. Publication Nos. 2003/0051263, 2003/0055020,2003/0056235, 2004/265839, 2005/0100913, 2006/0024798, 2008/0050342,2008/0081373, 2008/0248576, and 2008/055443; and/or U.S. Pat. Nos.6,506,559, 7,282,564, 7,538,095, and 7,560,438 (incorporated herein byreference). In some instances, the anti-CTLA-4 RNAi molecules take theform of double stranded RNAi molecules described in European Patent No.EP 1309726 (incorporated herein by reference). In some instances, theanti-CTLA-4 RNAi molecules take the form of double stranded RNAimolecules described in U.S. Pat. Nos. 7,056,704 and 7,078,196(incorporated herein by reference). In some embodiments, the CTLA-4inhibitor is an aptamer described in International Patent ApplicationPublication No. WO 2004/081021 (incorporated herein by reference).

In other embodiments, the anti-CTLA-4 RNAi molecules of the presentinvention are RNA molecules described in U.S. Pat. Nos. 5,898,031,6,107,094, 7,432,249, and 7,432,250, and European Application No. EP0928290 (incorporated herein by reference).

In some embodiments, the present invention includes a method of treatinga patient with a cancer comprising the steps of administering a TILregimen, wherein the TIL regimen includes a TIL product geneticallymodified to express a CCR, and further comprising the step ofadministering a CTLA-4 inhibitor. In some embodiments, the presentinvention includes a composition comprising (i) a TIL productgenetically modified to express a CCR and (ii) a CTLA-4 inhibitor. Insome embodiments, the present invention includes a kit comprising (i) aTIL product genetically modified to express a CCR and (ii) a CTLA-4inhibitor.

In some embodiments, the present invention includes a method of treatinga patient with a cancer comprising the steps of administering a TILregimen, wherein the TIL regimen includes a TIL product geneticallymodified to express a CCR, and further comprising the steps ofadministering a CTLA-4 inhibitor and either a PD-1 inhibitor or a PD-L1inhibitor. In some embodiments, the present invention includes acomposition comprising (i) a TIL product genetically modified to expressa CCR, (ii) a CTLA-4 inhibitor, and (iii) either a PD-1 inhibitor or aPD-L1 inhibitor. In some embodiments, the present invention includes akit comprising (i) a TIL product genetically modified to express a CCR,(ii) a CTLA-4 inhibitor, and (iii) either a PD-1 inhibitor or a PD-L1inhibitor.

4. Lymphodepletion Preconditioning of Patients

In an embodiment, the invention includes a method of treating a cancerwith a population of TILs, wherein a patient is pre-treated withnon-myeloablative chemotherapy prior to an infusion of TILs according tothe present disclosure. In an embodiment, the invention includes apopulation of TILs for use in the treatment of cancer in a patient whichhas been pre-treated with non-myeloablative chemotherapy. In anembodiment, the population of TILs is for administration by infusion. Inan embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) andfludarabine 25 mg/m2/d for 5 days (days 27 to 23 prior to TIL infusion).In an embodiment, after non-myeloablative chemotherapy and TIL infusion(at day 0) according to the present disclosure, the patient receives anintravenous infusion of IL-2 (aldesleukin, commercially available asPROLEUKIN) intravenously at 720,000 IU/kg every 8 hours to physiologictolerance. In certain embodiments, the population of TILs is for use intreating cancer in combination with IL-2, wherein the IL-2 isadministered after the population of TILs.

Experimental findings indicate that lymphodepletion prior to adoptivetransfer of tumor-specific T lymphocytes plays a key role in enhancingtreatment efficacy by eliminating regulatory T cells and competingelements of the immune system (‘cytokine sinks’). Accordingly, someembodiments of the invention utilize a lymphodepletion step (sometimesalso referred to as “immunosuppressive conditioning”) on the patientprior to the introduction of the TILs of the invention.

In general, lymphodepletion is achieved using administration offludarabine or cyclophosphamide (the active form being referred to asmafosfamide) and combinations thereof. Such methods are described inGassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski,et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J.Clin. Oncol. 2008, 26, 5233-5239, and Dudley, et al., J. Clin. Oncol.2005, 23, 2346-2357, all of which are incorporated by reference hereinin their entireties.

In some embodiments, the fludarabine is administered at a concentrationof 0.5 μg/mL to 10 μg/mL fludarabine. In some embodiments, thefludarabine is administered at a concentration of 1 μg/mL fludarabine.In some embodiments, the fludarabine treatment is administered for 1day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In someembodiments, the fludarabine is administered at a dosage of 10mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, thefludarabine treatment is administered for 2-7 days at 35 mg/kg/day. Insome embodiments, the fludarabine treatment is administered for 4-5 daysat 35 mg/kg/day. In some embodiments, the fludarabine treatment isadministered for 4-5 days at 25 mg/kg/day.

In some embodiments, the mafosfamide, the active form ofcyclophosphamide, is obtained at a concentration of 0.5 μg/mL-10 μg/mLby administration of cyclophosphamide. In some embodiments, mafosfamide,the active form of cyclophosphamide, is obtained at a concentration of 1μg/mL by administration of cyclophosphamide. In some embodiments, thecyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4days, 5 days, 6 days, or 7 days or more. In some embodiments, thecyclophosphamide is administered at a dosage of 100 mg/m2/day, 150mg/m2/day, 175 mg/m2/day, 200 mg/m2/day, 225 mg/m2/day, 250 mg/m2/day,275 mg/m2/day, or 300 mg/m2/day. In some embodiments, thecyclophosphamide is administered intravenously (i.e., i.v.) In someembodiments, the cyclophosphamide treatment is administered for 2-7 daysat 35 mg/kg/day. In some embodiments, the cyclophosphamide treatment isadministered for 4-5 days at 250 mg/m2/day i.v. In some embodiments, thecyclophosphamide treatment is administered for 4 days at 250 mg/m2/dayi.v.

In some embodiments, lymphodepletion is performed by administering thefludarabine and the cyclophosphamide together to a patient. In someembodiments, fludarabine is administered at 25 mg/m2/day i.v. andcyclophosphamide is administered at 250 mg/m2/day i.v. over 4 days.

In an embodiment, the lymphodepletion is performed by administration ofcyclophosphamide at a dose of 60 mg/m2/day for two days followed byadministration of fludarabine at a dose of 25 mg/m2/day for five days.

In an embodiment, the lymphodepletion is performed by administration ofcyclophosphamide at a dose of 60 mg/m2/day for two days andadministration of fludarabine at a dose of 25 mg/m2/day for five days,wherein cyclophosphamide and fludarabine are both administered on thefirst two days, and wherein the lymphodepletion is performed in fivedays in total.

In an embodiment, the lymphodepletion is performed by administration ofcyclophosphamide at a dose of about 50 mg/m2/day for two days andadministration of fludarabine at a dose of about 25 mg/m2/day for fivedays, wherein cyclophosphamide and fludarabine are both administered onthe first two days, and wherein the lymphodepletion is performed in fivedays in total.

In an embodiment, the lymphodepletion is performed by administration ofcyclophosphamide at a dose of about 50 mg/m2/day for two days andadministration of fludarabine at a dose of about 20 mg/m2/day for fivedays, wherein cyclophosphamide and fludarabine are both administered onthe first two days, and wherein the lymphodepletion is performed in fivedays in total.

In an embodiment, the lymphodepletion is performed by administration ofcyclophosphamide at a dose of about 40 mg/m2/day for two days andadministration of fludarabine at a dose of about 20 mg/m2/day for fivedays, wherein cyclophosphamide and fludarabine are both administered onthe first two days, and wherein the lymphodepletion is performed in fivedays in total.

In an embodiment, the lymphodepletion is performed by administration ofcyclophosphamide at a dose of about 40 mg/m2/day for two days andadministration of fludarabine at a dose of about 15 mg/m2/day for fivedays, wherein cyclophosphamide and fludarabine are both administered onthe first two days, and wherein the lymphodepletion is performed in fivedays in total.

In an embodiment, the lymphodepletion is performed by administration ofcyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of25 mg/m2/day for two days followed by administration of fludarabine at adose of 25 mg/m2/day for three days.

In an embodiment, the cyclophosphamide is administered with mesna. In anembodiment, mesna is administered at 15 mg/kg. In an embodiment wheremesna is infused, and if infused continuously, mesna can be infused overapproximately 2 hours with cyclophosphamide (on Days −5 and/or −4), thenat a rate of 3 mg/kg/hour for the remaining 22 hours over the 24 hoursstarting concomitantly with each cyclophosphamide dose.

In an embodiment, the lymphodepletion comprises the step of treating thepatient with an IL-2 regimen starting on the day after administration ofthe third population of TILs to the patient.

In an embodiment, the lymphodepletion comprises the step of treating thepatient with an IL-2 regimen starting on the same day as administrationof the third population of TILs to the patient.

In some embodiments, the lymphodeplete comprises 5 days ofpreconditioning treatment. In some embodiments, the days are indicatedas days −5 through −1, or Day 0 through Day 4. In some embodiments, theregimen comprises cyclophosphamide on days −5 and −4 (i.e., days 0 and1). In some embodiments, the regimen comprises intravenouscyclophosphamide on days −5 and −4 (i.e., days 0 and 1). In someembodiments, the regimen comprises 60 mg/kg intravenous cyclophosphamideon days −5 and −4 (i.e., days 0 and 1). In some embodiments, thecyclophosphamide is administered with mesna. In some embodiments, theregimen further comprises fludarabine. In some embodiments, the regimenfurther comprises intravenous fludarabine. In some embodiments, theregimen further comprises 25 mg/m2 intravenous fludarabine. In someembodiments, the regimen further comprises 25 mg/m2 intravenousfludarabine on days −5 and −1 (i.e., days 0 through 4). In someembodiments, the regimen further comprises 25 mg/m2 intravenousfludarabine on days −5 and −1 (i.e., days 0 through 4).

In some embodiments, the non-myeloablative lymphodepletion regimencomprises the steps of administration of cyclophosphamide at a dose of60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two daysfollowed by administration of fludarabine at a dose of 25 mg/m2/day forfive days.

In some embodiments, the non-myeloablative lymphodepletion regimencomprises the steps of administration of cyclophosphamide at a dose of60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two daysfollowed by administration of fludarabine at a dose of 25 mg/m2/day forthree days.

In some embodiments, the non-myeloablative lymphodepletion regimen isadministered according to Table 26.

TABLE 26 Exemplary lymphodepletion and treatment regimen. Day −5 −4 −3−2 −1 0 1 2 3 4 Cyclophosphamide X X 60 mg/kg Mesna (as needed) X XFludarabine 25 X X X X X mg/m²/day TIL infusion X

In some embodiments, the non-myeloablative lymphodepletion regimen isadministered according to Table 27.

TABLE 27 Exemplary lymphodepletion and treatment regimen. Day −4 −3 −2−1 0 1 2 3 4 Cyclophosphamide 60 mg/kg X X Mesna (as needed) X XFludarabine 25 mg/m²/day X X X X TIL infusion X

In some embodiments, the non-myeloablative lymphodepletion regimen isadministered according to Table 28.

TABLE 28 Exemplary lymphodepletion and treatment regimen. Day −3 −2 −1 01 2 3 4 Cyclophosphamide 60 mg/kg X X Mesna (as needed) X X Fludarabine25 mg/m²/day X X X TIL infusion X

In some embodiments, the non-myeloablative lymphodepletion regimen isadministered according to Table 29.

TABLE 29 Exemplary lymphodepletion and treatment regimen. Day −5 −4 −3−2 −1 0 1 2 3 4 Cyclophosphamide X X 60 mg/kg Mesna (as needed) X XFludarabine 25 X X X mg/m²/day TIL infusion X

In some embodiments, the non-myeloablative lymphodepletion regimen isadministered according to Table 30.

TABLE 30 Exemplary lymphodepletion and treatment regimen. Day −5 −4 −3−2 −1 0 1 2 3 4 Cyclophosphamide X X 300 mg/kg Mesna (as needed) X XFludarabine 30 X X X X X mg/m²/day TIL infusion X

In some embodiments, the non-myeloablative lymphodepletion regimen isadministered according to Table 31.

TABLE 31 Exemplary lymphodepletion and treatment regimen. Day −4 −3 −2−1 0 1 2 3 4 Cyclophosphamide 300 mg/kg X X Mesna (as needed) X XFludarabine 30 mg/m²/day X X X X TIL infusion X

In some embodiments, the non-myeloablative lymphodepletion regimen isadministered according to Table 32.

TABLE 32 Exemplary lymphodepletion and treatment regimen. Day −3 −2 −1 01 2 3 4 Cyclophosphamide 300 mg/kg X X Mesna (as needed) X X Fludarabine30 mg/m²/day X X X TIL infusion X

In some embodiments, the non-myeloablative lymphodepletion regimen isadministered according to Table 33.

TABLE 33 Exemplary lymphodepletion and treatment regimen. Day −5 −4 −3−2 −1 0 1 2 3 4 Cyclophosphamide X X 300 mg/kg Mesna (as needed) X XFludarabine 30 X X X mg/m²/day TIL infusion X

In some embodiments, the TIL infusion used with the foregoingembodiments of myeloablative lymphodepletion regimens may be any TILcomposition described herein, including TIL products geneticallymodified to express a CCR as described herein, and may also includeinfusions of MILs and PBLs in place of the TIL infusion, as well as theaddition of IL-2 regimens and administration of co-therapies (such asPD-1 and PD-L1 inhibitors) as described herein.

5. IL-2 Regimens

In an embodiment, the IL-2 regimen comprises a high-dose IL-2 regimen,wherein the high-dose IL-2 regimen comprises aldesleukin, or abiosimilar or variant thereof, administered intravenously starting onthe day after administering a therapeutically effective portion oftherapeutic population of TILs, wherein the aldesleukin or a biosimilaror variant thereof is administered at a dose of 0.037 mg/kg or 0.044mg/kg IU/kg (patient body mass) using 15-minute bolus intravenousinfusions every eight hours until tolerance, for a maximum of 14 doses.Following 9 days of rest, this schedule may be repeated for another 14doses, for a maximum of 28 doses in total. In some embodiments, IL-2 isadministered in 1, 2, 3, 4, 5, or 6 doses. In some embodiments, IL-2 isadministered at a maximum dosage of up to 6 doses.

In an embodiment, the IL-2 regimen comprises a decrescendo IL-2 regimen.Decrescendo IL-2 regimens have been described in O'Day, et al., J. Clin.Oncol. 1999, 17, 2752-61 and Eton, et al., Cancer 2000, 88, 1703-9, thedisclosures of which are incorporated herein by reference. In anembodiment, a decrescendo IL-2 regimen comprises 18×106 IU/m2aldesleukin, or a biosimilar or variant thereof, administeredintravenously over 6 hours, followed by 18×106 IU/m2 administeredintravenously over 12 hours, followed by 18×106 IU/m2 administeredintravenously over 24 hours, followed by 4.5×106 IU/m2 administeredintravenously over 72 hours. This treatment cycle may be repeated every28 days for a maximum of four cycles. In an embodiment, a decrescendoIL-2 regimen comprises 18,000,000 IU/m2 on day 1, 9,000,000 IU/m2 on day2, and 4,500,000 IU/m2 on days 3 and 4.

In an embodiment, the IL-2 regimen comprises a low-dose IL-2 regimen.Any low-dose IL-2 regimen known in the art may be used, including thelow-dose IL-2 regimens described in Dominguez-Villar and Hafler, Nat.Immunology 2000, 19, 665-673; Hartemann, et al., Lancet DiabetesEndocrinol. 2013, 1, 295-305; and Rosenzwaig, et al., Ann. Rheum. Dis.2019, 78, 209-217, the disclosures of which are incorporated herein byreference. In an embodiment, a low-dose IL-2 regimen comprises 18×106 IUper m2 of aldesleukin, or a biosimilar or variant thereof, per 24 hours,administered as a continuous infusion for 5 days, followed by 2-6 dayswithout IL-2 therapy, optionally followed by an additional 5 days ofintravenous aldesleukin or a biosimilar or variant thereof, as acontinuous infusion of 18×106 IU per m2 per 24 hours, optionallyfollowed by 3 weeks without IL-2 therapy, after which additional cyclesmay be administered.

In an embodiment, the IL-2 regimen comprises administration of pegylatedIL-2 every 1, 2, 4, 6, 7, 14 or 21 days at a dose of 0.10 mg/day to 50mg/day. In an embodiment, the IL-2 regimen comprises administration ofbempegaldesleukin, or a fragment, variant, or biosimilar thereof, every1, 2, 4, 6, 7, 14 or 21 days at a dose of 0.10 mg/day to 50 mg/day.

In an embodiment, the IL-2 regimen comprises administration of THOR-707,or a fragment, variant, or biosimilar thereof, every 1, 2, 4, 6, 7, 14or 21 days at a dose of 0.10 mg/day to 50 mg/day.

In an embodiment, the IL-2 regimen comprises administration ofnemvaleukin alfa, or a fragment, variant, or biosimilar thereof, every1, 2, 4, 6, 7, 14 or 21 days at a dose of 0.10 mg/day to 50 mg/day.

In an embodiment, the IL-2 regimen comprises administration of an IL-2fragment engrafted onto an antibody backbone. In an embodiment, the IL-2regimen comprises administration of an antibody-cytokine engraftedprotein that binds the IL-2 low affinity receptor. In an embodiment, theantibody cytokine engrafted protein comprises a heavy chain variableregion (VH), comprising complementarity determining regions HCDR1,HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1,LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted intoa CDR of the VH or the VL, wherein the antibody cytokine engraftedprotein preferentially expands T effector cells over regulatory T cells.In an embodiment, the antibody cytokine engrafted protein comprises aheavy chain variable region (VH), comprising complementarity determiningregions HCDR1, HCDR2, HCDR3; a light chain variable region (VL),comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragmentthereof engrafted into a CDR of the VH or the VL, wherein the IL-2molecule is a mutein, and wherein the antibody cytokine engraftedprotein preferentially expands T effector cells over regulatory T cells.In an embodiment, the IL-2 regimen comprises administration of anantibody comprising a heavy chain selected from the group consisting ofSEQ ID NO:29 and SEQ ID NO:38 and a light chain selected from the groupconsisting of SEQ ID NO:37 and SEQ ID NO:39, or a fragment, variant, orbiosimilar thereof, every 1, 2, 4, 6, 7, 14 or 21 days at a dose of 0.10mg/day to 50 mg/day.

In some embodiments, the antibody cytokine engrafted protein describedherein has a longer serum half-life that a wild-type IL-2 molecule suchas, but not limited to, aldesleukin (Proleukin®) or a comparablemolecule.

In some embodiments, the TIL infusion used with the foregoingembodiments of myeloablative lymphodepletion regimens may be any TILcomposition described herein and may also include infusions of MILs andPBLs in place of the TIL infusion, as well as the addition of IL-2regimens and administration of co-therapies (such as PD-1 and PD-L1inhibitors) as described herein.

In some embodiments, the present invention includes a method of treatinga patient with a cancer comprising the step of administering a TILregimen, wherein the TIL regimen includes a TIL product geneticallymodified to express a CCR, and further comprising the step ofadministering an IL-2 regimen. In some embodiments, the presentinvention includes a composition comprising (i) a TIL productgenetically modified to express a CCR and (ii) an IL-2 regimen. In someembodiments, the present invention includes a kit comprising (i) a TILproduct genetically modified to express a CCR and (ii) an IL-2 regimen.

In some embodiments, the present invention includes a method of treatinga patient with a cancer comprising the steps of administering a TILregimen, wherein the TIL regimen includes a TIL product geneticallymodified to express a CCR, and further comprising the steps ofadministering an IL-2 regimen and either a PD-1 inhibitor or a PD-L1inhibitor. In some embodiments, the present invention includes acomposition comprising (i) a TIL product genetically modified to expressa CCR, (ii) an IL-2 regimen, and (iii) either a PD-1 inhibitor or aPD-L1 inhibitor. In some embodiments, the present invention includes akit comprising (i) a TIL product genetically modified to express a CCR,(ii) an IL-2 regimen, and (iii) either a PD-1 inhibitor or a PD-L1inhibitor.

In some embodiments, the present invention includes a method of treatinga patient with a cancer comprising the steps of administering a TILregimen, wherein the TIL regimen includes a TIL product geneticallymodified to express a CCR, and further comprising the steps ofadministering a CTLA-4 inhibitor and an IL-2 regimen. In someembodiments, the present invention includes a composition comprising (i)a TIL product genetically modified to express a CCR, (ii) a CTLA-4inhibitor, and (iii) an IL-2 regimen. In some embodiments, the presentinvention includes a kit comprising (i) a TIL product geneticallymodified to express a CCR, (ii) a CTLA-4 inhibitor, and (iii) an IL-2regimen.

In some embodiments, the present invention includes a method of treatinga patient with a cancer comprising the steps of administering a TILregimen, wherein the TIL regimen includes a TIL product geneticallymodified to express a CCR, and further comprising the steps ofadministering an IL-2 regimen, a CTLA-4 inhibitor, and either a PD-1inhibitor or a PD-L1 inhibitor. In some embodiments, the presentinvention includes a composition comprising (i) a TIL productgenetically modified to express a CCR, (ii) an IL-2 regimen, (iii)either a PD-1 inhibitor or a PD-L1 inhibitor, and (iv) a CTLA-4inhibitor. In some embodiments, the present invention includes a kitcomprising (i) a TIL product genetically modified to express a CCR, (ii)an IL-2 regimen, (iii) either a PD-1 inhibitor or a PD-L1 inhibitor, and(iv) a CTLA-4 inhibitor.

B. Adoptive Cell Transfer

Adoptive cell transfer (ACT) is an effective form of immunotherapy andinvolves the transfer of immune cells with antitumor activity intocancer patients. ACT is a treatment approach that involves theidentification, in vitro, of lymphocytes with antitumor activity, the invitro expansion of these cells to large numbers and their infusion intothe cancer-bearing host. Lymphocytes used for adoptive transfer can bederived from the stroma of resected tumors (tumor infiltratinglymphocytes or TILs). TILs for ACT can be prepared as described herein.In some embodiments, the TILs are prepared, for example, according to amethod as described in FIG. 2A and/or FIG. 9 . They can also be derivedor from blood if they are genetically engineered to express antitumorT-cell receptors (TCRs) or chimeric antigen receptors (CARs), enrichedwith mixed lymphocyte tumor cell cultures (MLTCs), or cloned usingautologous antigen presenting cells and tumor derived peptides. ACT inwhich the lymphocytes originate from the cancer-bearing host to beinfused is termed autologous ACT. U.S. Publication No. 2011/0052530relates to a method for performing adoptive cell therapy to promotecancer regression, primarily for treatment of patients suffering frommetastatic melanoma, which is incorporated by reference in its entiretyfor these methods. In some embodiments, TILs can be administered asdescribed herein. In some embodiments, TILs can be administered in asingle dose. Such administration may be by injection, e.g., intravenousinjection. In some embodiments, TILs and/or cytotoxic lymphocytes may beadministered in multiple doses. Dosing may be once, twice, three times,four times, five times, six times, or more than six times per year.Dosing may be once a month, once every two weeks, once a week, or onceevery other day. Administration of TILs and/or cytotoxic lymphocytes maycontinue as long as necessary.

V. Coordinating Manufacturing of Cell Expansion Product and PatientTreatment Events

As discussed herein, the timing from tumor resection from the patient tocompletion of the TIL manufacturing varies depending on several factorsincluding, for example, the size of the tumor obtainable from thepatient, cell count at the end of various stage of manufacturing, numberof days for which various stages of manufacturing are performed, etc. Asa consequence, certain amount of flexibility is needed in schedulingvarious patient treatment events. In some embodiments, patient treatmentevent includes lymphodepletion. In some embodiments, patient treatmentevent includes TIL infusion. In some embodiments, the patient treatmentevent includes a leukapheresis procedure. In some embodiments, patienttreatment event includes administration of an IL-2 regimen. In someembodiments, patient treatment event includes tumor resection. In someembodiments, patient treatment event includes inpatient stay forpost-procedure treatments.

An aspect of the present disclosure provides for a system forimplementing the methods disclosed herein. The system may include apatient portal, a manufacturing portal, a clinician portal and alogistics portal. The patient portal enables a patient or a personassociated with the patient (e.g., a caregiver, a guardian or a legallyauthorized agent) (together referred to herein as “the patient” forconvenient reference) can access the information relating to theschedule of the patient treatment events. The patient portal may requirethe patient to provide authentication information to access thisinformation. The patient portal may also allow the patient to editinformation relating to the patient such as, for example, address orother personal information.

The clinician portal, also referred to as the hospital-side interface,enables the clinic (i.e., the clinic personnel) to access and/or editinformation relating to the patient, the manufacturing process, themanufacturing facilities, the logistics provider as well as theinformation relating to various patient treatment events performed or tobe performed at the clinic.

The manufacturing portal communicates with the hospital-side interfaceand the logistics portal/logistics interface to determine and conveyinformation relating to the manufacturing process and/or availability ofthe manufacturing slots. The manufacturing portal also enablesgeneration of manufacturing labels including updated quality informationat various manufacturing steps as disclosed herein.

The logistics portal, also referred to herein as the logisticsinterface, communicates with the manufacturing portal and thehospital-side interface exchange information relating to the schedulefor shipping of material (i.e., the solid tumor, or the manufacturedcell therapy product) between the clinic and the manufacturing facilityincluding, for example, the manufacturing schedule, the availability ofmanufacturing slots and the schedule of patient treatment events tofacilitate timely shipment of the material.

The system for implemented the various methods disclosed herein can beimplemented on a computing system by either implementing a proprietarycomputer program or by suitably modifying a commercially availablesoftware platform such as, for example, one provided by Vineti,Salesforce.com, Salesforce Health Cloud, IQVIA, TracSel, and SAP.

The modification of commercially available software platform so as tosuitably implement the system and perform one or more methods disclosedherein may cause the platform to perform processes that it was notoriginally designed for. In other words, the modification, in someembodiments, may be based on unconventional use of the various toolsprovided by the platform. In some embodiments, the system forimplementation of various methods disclosed herein can include asoftware platform such as a framework program that integrates anenterprise resource planning (ERP) system that enables automation oflogistical tasks and a manufacturing execution system (MES) that enablesautomation of manufacturing tasks. An example of such a frameworkprogram is described in U.S. Pat. No. 7,343,605, which is incorporatedherein by reference in its entirety.

For example, alongside IVIES applications, applications from theenterprise control level (Enterprise Resource Planning level) and fromthe automation level (controls level) can also be integrated via aframework program and monitored or managed via a workstation (e.g., atthe clinical facility, at the manufacturing facility or at the courierfacility). The framework program may thus form an integration platformfor the entire patient treatment process including registering thepatient, procurement of the tumor, shipping the tumor to themanufacturing facility, manufacturing the therapeutic or expanded celltherapy product, shipping the therapeutic cell therapy product to theclinical facility, administering the therapy to the patient, andsubsequent patient treatment events. Different applications from theenterprise control level, the MES level and the automation level can beintegrated by the framework program simply and cost-effectively with theaid of adapters and/or wrappers. The framework program must therefore beregarded as a middleware platform and as a manufacturing applicationintegration tool. An end user (e.g. a case manager) can see therespective status of the applications to be monitored via a workstationand can also access data and methods of the applications. Further theend user can connect applications to each other by means of this access.

The framework program therefore makes it possible firstly to achieve avertical integration of applications from different enterprise levelsand secondly the framework program enables a horizontal integration ofapplications on the MES level.

For instance, a software-based case object, that allows storage ofinformation in the platform, is the overall definition of the type ofinformation being stored. For example, a software-based case object inthe off-the-shelf platform allows storage of information regardingcustomer inquiries. For such object, there may be multiple records thatstore the information about specific instances of that type of data.Thus, a case record to store the information about an individual'straining inquiry and another case record to store the information aboutanother individual's configuration issue. These objects may becustomized to store information relating to, e.g., a patient, thepatient treatment events for that patient and a corresponding schedule.

Similarly, automated actions such as notifications and alerts, andworkflow rules such as business logic actions, provided within theoff-the-shelf platform may be modified to trigger changes in schedulesand process steps such as, e.g., changes in manufacturing processes andthe consequent changes in manufacturing schedules, availability ofmanufacturing slots and schedules of patient treatment events based onresults of a QA test.

In some embodiments, the system disclosed herein provides qualityassurance features for determining probable points of failure during themanufacturing process such as those described in U.S. Pat. Nos.10,747,209, and 7,799,273, which is incorporated herein by reference inits entirety.

In some embodiments, the system disclosed herein provides data systemsconfigured to maintain integrity of electronic batch records createdduring the process of manufacturing a final cell therapy product from asolid tumor (or a fragment thereof) obtained from the patient such asthose described in U.S. Pat. No. 8,491,839, which is incorporated hereinby reference in its entirety. In some embodiments, the system disclosedherein includes an application software adapted for use in cellmanufacturing process wherein the cell manufacturing process producesexpanded cell therapy product such as a therapeutic population ofT-cells. The application software is configured to control a pluralityof devices included in a closed system for cell manufacturing such asgas permeable devices.

In some embodiments, the system disclosed herein integrates applicationsoftware and cell manufacturing methods disclosed herein to provide acomprehensive validation and quality assurance protocol that is used bya plurality of end users whereby the data compiled from the system isanalyzed and used to determine is quality assurance protocols andvalidation protocols are being achieved.

In some embodiments, the system applies the application software tomultiple product lines and/or multiple cell manufacturing facilities,whereby multiple cell manufacturing lines and multiple product lines aremonitored and controlled using the same system.

In some embodiments, the system implements the methods described hereininto the batch optimization of cell culture systems of the cellmanufacturing process whereby the data compiled by the cell culturesystem is tracked continuously overtime and the data is used to analyzethe cell culture system. In addition, the said data is integrated andused to analyze the quality control process of the cell manufacturingprocess at-large.

In some embodiments, the system disclosed herein enables design of workstations for performing various steps of the process of manufacturing afinal cell therapy product. The work stations may be configured toenable verification of data associated with a previous step to ensureintegrity of the process being performed and enable a remedial action,where possible. Examples of such work station designs are provided inU.S. Pat. No. 8,041,444, which is incorporated herein by reference inits entirety.

In some embodiments, the system disclosed herein enables coordinationbetween a hospital facility and various manufacturers capable ofmanufacturing the cell therapy product from the solid tumor obtained atthe hospital facility by providing access to available manufacturingschedules at the various manufacturers. Examples of systems enablingsuch coordination are provided in U.S. Pat. No. 8,069,071, which isincorporated herein by reference in its entirety.

In some embodiments, the system disclosed herein is based on a cloudbased architecture which enables various entities associated with themanufacturing of the cell therapy product real-time access (withappropriate permissions) to information relating to the manufacturingand transportation of the cell therapy product. Examples of suchcloud-based architecture are provided in U.S. Pat. No. 9,965,562, whichis incorporated herein by reference in its entirety.

In some embodiments, the system disclosed herein enables personnelassociated with obtaining the tumor from the patient, manufacturing ofthe cell therapy product from the obtained tumor and transportation ofthe tumor and the cell therapy product, to identify and authenticatethemselves using electronic signatures for process control and approval,such as described in US Patent Application Publication No. 2004/0243260,which is incorporated herein by reference in its entirety.

In some embodiments, the system may further include, or incorporatetherein, a customer relationship management (CRM) subsystem. The CRMsubsystem may archive information relating to various parties such as,for example, doctors, hospital sites, nurses, billers, businesscontacts, etc. The information is maintained with the CRM subsystem toenable maintenance of a single view of parties in one unified funnel,allowing sharing of platform-based, single-funnel views of prospects andparties in a privacy-compliant manner within the system. Informationrelating to relationship between parties can be shared in a distributedmulti-master, multi-slave or peer-peer context, partially or fullyanonymously by removal of personally identifiable information, to one ormore distributed slave or peer CRM systems. One example of such a CRMsubsystem is disclosed in US Patent Application Publication No.2014/0244351, U.S. Pat. Nos. 8,489,451, and 9,342,292, each of which isincorporated herein by reference in its entirety.

In some embodiments, the system described herein may be operable toprovide a network that includes a central event processing subsystem forreceiving, processing, and routing messages triggered by real-timemedical events and/or manufacturing events. The central event processingsystem, for example, may identify patient information associated with amedical event and match the patient information to one or more of ahealth plan, a clinical location, a TIL manufacturing facility, and/or alogistics provider. Upon matching the medical and/or manufacturing eventwith the interested parties, the central event processing subsystem mayforward at least a portion of the information regarding the medicaland/or manufacturing event to one or more interested parties within ashort period of time of the triggering event (e.g., in near real-time).For example, based upon rules associated with each interested party, thecentral event processing system may forward information and/or issue analert or notification to an interested party to make the interestedparty aware of the medical event. Example of such a central eventprocessing subsystem is described in US Patent Application PublicationNo. 2014/0372147, and U.S. Pat. No. 10,242,060, each of which isincorporated herein by reference in its entirety.

In some embodiments, the system disclosed herein is operable to select aworkflow for a cancer treatment regimen including patient treatmentevents such as, for example, tumor resection, lymphodelpetion,leukapheresis, infusion of TILs, and/or IL-2 regimen, or other patienttreatment events disclosed herein. Upon selection of the workflow, thesystem may produce purchase order corresponding to the cell infusiontherapy on behalf of a patient, wherein the order corresponding to thecell infusion therapy includes at least one of a cell order request anda request for specified treatment regimen corresponding to the cancertreatment. Examples of such workflow selection subsystem are disclosedin US Patent Application Publication No. US 2014/0088985, which isincorporated herein by reference in its entirety.

In some embodiments, the system may include a telephony subsystem thatmay include authentication of service requests including authenticationof a remote access device prior to text or audio communication with apatient or a representative of the patient. In some embodiments, theauthentication may be accomplished by automatically authenticating theremove access device or by asking questions to the patient (or therepresentative). Example of the such a telephony subsystem andauthentication method is disclosed in US Patent Application PublicationNo. US 2019/0026747, which is incorporated herein by reference in itsentirety.

A. System for Coordinating Manufacturing of Cell Expansion Product

Embodiments of the present disclosure include a method and a system forcoordinating the manufacturing of a cell therapy product such as, forexample, T-cells or TILs for a patient, and dynamically schedulingvarious stages of the manufacturing process as well as various patienttreatment events based on the progress and success of various stages ofthe manufacturing process.

The methods and systems described herein are further operable to providethe specific technical advantage over existing systems of providing acontinuous and automatic chain of custody and chain of identity for apatient-specific biological sample during an immunotherapy procedure, tocreate a computerized information portal that interested parties—such asthe patient, physician, manufacturer, and other medical personnel—mayuse to quickly understand and track the current phase of theimmunotherapy procedure and the status of the patient's biologicalsample during the procedure. A lack of ability to maintain a chain ofcustody and chain of identity—resulting in delays during themanufacturing process which, for a patient dealing with alife-threatening illness, may be immeasurably severe.

Embodiments of the present disclosure enable maintenance of the chain ofcustody of the biological material during the manufacturing process(including QA, manufacturing, release testing, and finalizing forshipment), and the chain of custody of the biological material duringthe final product delivery process (including shipment and delivery tothe infusion site). Embodiments of the present disclosure further enablecontinuously and constantly associating chain of custody with thespecific patient—thereby ensuring a complete chain of identity betweenthe patient and the biological material during all phases ofmanufacturing.

The maintenance of COC and COI is performed by associating each eventduring the entire journey of the biological material from the patientthrough transportation, the manufacturing process, transportation andback to the patient with a cell order identifier and a patient-specificidentifier (that is unique to the patient), and tracking each eventduring the entire journey.

In addition, the methods and systems described herein are operable tointegrate and synchronize obtaining the living tissue from the patientwith the manufacturing process, as well as provide capability forauditing each step from obtaining the living tissue to administering ofthe cell therapy product and subsequent treatment events for COC andCOI.

The methods and systems described herein are further operable forcoordinating logistics for obtaining the living tissue from the patient,delivery of the living tissue to a selected manufacturing facility,manufacturing of the cell therapy product at the selected manufacturingfacility, and delivery of the cell therapy product from themanufacturing facility to the clinic for patient treatment.

FIG. 3A shows a block diagram for a system for coordinating themanufacturing of a cell therapy product for a patient in accordance withan embodiment of the present disclosure. In some embodiments,functionality performed by the components in FIG. 3A may be integratedinto a single component. Also, in some embodiments, functionalityperformed by one or more components in FIG. 3A may also be performed byother components in FIG. 3A.

Referring to FIG. 3A, the system 300 may include a hospital-sideinterface 110, an events scheduler 120, a logistics interface (alsoreferred to herein as the courier portal) 130 and/or a manufacturingportal 140. Each of the hospital-side interface 110, the eventsscheduler 120, the logistics interface 130 and the manufacturing portal140 communicates with the other three, e.g., using a communicationnetwork such as a LAN, a WAN (e.g., the Internet), and/or a cellularnetwork.

In some embodiments, the hospital-side interface 110, the eventsscheduler 120, the logistics interface 130, the manufacturing portal140, and corresponding modules (shown, e.g., in FIG. 3 ) suitably modifyor build upon commercially available software platforms, such as, forexample, those provided by Vineti, Salesforce.com, Salesforce HealthCloud, IQVIA, TracSel, and SAP.

The hospital-side interface 110 may be associated with or may interactwith a hospital or other facility responsible for administration of atreating a patient, e.g., providing a therapy for cancer as disclosedherein. In some embodiments, the hospital-side interface 110 isassociated with a clinical facility that performs a procedure forobtaining a solid tumor from a patient, and obtaining cell therapyproduct from the solid tumor or fragments thereof. In some embodiments,the clinical facility functions only to obtain the solid tumor while theprocess of obtaining the cell therapy product from the solid tumor orfragments thereof can be performed at a manufacturing facility.

In some embodiments, the hospital-side interface 110 is associated witha hospital that performs the infusion of an expanded cell therapyproduct obtained from a manufacturing facility and provides subsequentcare and/or any prior or follow-on treatment to the patient. Cliniciansor employees of the hospital or clinic may interact with the system 300via the hospital-side interface 110.

In some embodiments, the hospital-side interface 110 includes one ormore computing devices such as, for example, a desktop computer, a cloudserver, a laptop computer, mobile devices, or any other computing deviceincluding hardware and software modules that execute on a processor andinteract with a memory. The hospital-side interface 110 may beconnected, via wired or wireless connection, to computing devicesoperated at, or associated with, the hospital or clinical facility.

In some embodiments, the hospital-side interface 110 may include atracking module 112 configured for tracking the biological material(e.g., tumor from the patient, T-cells obtained from the patient,expanded T-cells at various stages of T-cell expansion process, etc.)from the patient from the time of extraction from the patient till thetime of infusion into the patient.

In some embodiments, when a patient is enrolled for a TIL infusiontreatment, a cell order request for manufacture of an expanded celltherapy product for the patient is created and a cell order identifierassociated with the cell order request is generated. In someembodiments, a purchase order is generated in accordance with the cellorder request and uploaded to the system from the hospital facility. Insome embodiments, the system may include an interface for generatingand/or uploading the purchase order.

In addition a patient-specific identifier unique to the patient isgenerated and associated with the cell order identifier. In someembodiments, the patient-specific identifier may include, for example, apatient ID # with sequential suffix, where the suffix is a single letteradded in sequence per patient (A-Z). The patient-specific identifier isunique in the system. In some embodiments, the patient-specificidentifier may be visible only to certain personas (described in detailelsewhere herein). In some embodiments, the patient-specific identifieris printed on every label printed through the journey of the patientbiological material, as well as the manufactured biological material. Insome embodiments, the patient-specific identifier is read only to allsystem users. In other words, the patient-specific identifier, in someembodiments, cannot be edited once generated.

In some embodiments, the cell order identifier may include informationsuch as, for example, a unique patient identifier, a cell orderidentifier, an order code, a cell order lot number, values of one ormore acceptance parameters at various time points, one or moreindicators indicating whether acceptance criteria are met at varioustime points, or a combination thereof.

In some embodiments, the cell order identifier and the patient-specificidentifier are scanned at each point where any biological material(e.g., solid tumor extracted from the patient, fragments thereof, celltherapy product extracted therefrom, or expanded cell therapy productobtained from expanding the cell therapy product) associated with thepatient changes custody or undergoes processing. Each scanned may belogged and verified by the tracking module 112 during the entire processfrom the time of extraction from the patient till the time of infusioninto the patient. The verification of patient-specific identifier ateach step during the entire process ensures a chain of identity (COI)for the product, while the verification of the cell order identifier ateach step during the entire process ensures a chain of custody (COC) forthe product. The details relating to maintenance of COI and COC aredescribed elsewhere herein.

In some embodiments, the scanned information is verified with thepurchase order generated by the hospital facility. For example, when ashipment is received at the manufacturing facility, the label on theshipping container may be scanned and the information on the label maybe verified with the purchase order to ensure accuracy. In someembodiments, the result of the verification is logged within the system.

It will be appreciated that while the tracking module 112 is shown inFIG. 3A, and described herein as being part of the hospital-sideinterface 110, the tracking module 112 can be implemented as astandalone computing device having a processor and a memory in someembodiments Similarly, the tracking module 112 can also be implementedas a standalone software module (e.g., stored on a cloud server) in someembodiments.

In some embodiments, the hospital-side interface 110 may provide limitedaccess to a patient, via a patient access module 116, to enable thepatient to obtain information relating to treatment procedures andcorresponding schedules. The patient access module 116 may also enablethe patient to provide information relating to oneself such as, forexample, personal identifying information, insurance information, andinformation relating to one's health condition for clinicians.

The hospital-side interface 110 may further include a procurement module114 operable to enable personnel at the clinical facility to obtain thesolid tumor from the patient in accordance with a predeterminedprotocol, and enter information relating to the procedure for obtainingthe solid tumor from the patient. The information relating to theprocedure for obtaining the solid tumor may, in some embodiments, bearchived to enable post-facto audit to ensure compliance with regulatoryrequirements.

In some embodiments, the procurement module 114 may further enable thepersonnel at the clinical facility to provide information to themanufacturing facility about the solid tumor and the processes used toobtain the solid tumor from the patient. In embodiments where theclinical facility is also operable to obtain cell therapy product fromthe solid tumor, the procurement module 114 may enable the personnel atclinical facility to provide information about the obtained cell therapyproduct such as, for example, the process or procedure used forobtaining the cell therapy product, and the results of quality controlassays performed on the obtained cell therapy product to themanufacturing facility.

In some embodiments, the hospital-side interface 110 may further includea manufacturing coordination module 118. The manufacturing coordinationmodule 118 is operable to enable users at the clinical facility toobtain information relating to one or more manufacturing facilities suchas, for example, availability of manufacturing slots and capabilitiesavailable at each of the one or more manufacturing facilities. Themanufacturing coordination module 118 further enables users at theclinical facility to coordinate the reservation of a manufacturing slotat a selected manufacturing facility, and coordinate with a logisticsprovider via the logistics interface 130 to arrange for transportationof the solid tumor, fragments thereof or cell therapy product obtainedfrom the patient. The manufacturing coordination module 118 furtherenables users at the clinical facility to obtain information relating tothe manufacturing process and/or quality control assays performed duringthe manufacturing process via the manufacturing portal 140.

The events scheduler 120 is operable to coordinate the scheduling ofvarious activities performed at the clinical facility and themanufacturing facility. Additionally or alternately, the eventsscheduler 120 may provide the information relating to scheduling ofvarious activities to a logistics provider so as to facilitatetransportation of the cell therapy product between the clinical facilityand the manufacturing facility. the facility for manufacturing the celltherapy product, e.g., facility where the cell therapy product expansiontakes place. As described in more detail below, based on differentevents that occur during the manufacturing of the cell therapy product,the event scheduler is configured to dynamically schedule when otherevent(s) should occur.

In some embodiments, the events scheduler 120 includes a computingdevice 122 such as, for example, a desktop computer, a server, a laptopcomputer, mobile devices, or any other computing device includinghardware and software modules that execute on a processor and interactwith a memory. The hardware and/or software modules include anacceptance determining module 123 and a scheduling module 125. In someembodiments, the computing device 122 may be a cloud server accessiblevia the hospital interface 110, the manufacturing portal 140 and thelogistics interface 130. The events scheduler 120 may be connected, viawired or wireless connection, to computing devices operated at, orassociated with, the manufacturing facility and/or the hospitalfacility.

It will be appreciated that while the acceptance determination module123 is shown in FIG. 3A as being part of the events scheduler 120, theacceptance determination module 123 may also be implemented as part ofthe manufacturing portal 140. Alternately, the acceptance determinationmodule 123 may also be implemented as a standalone software module,e.g., hosted on a cloud server.

The logistics interface or courier portal 130 may be associated with ormay interact with a logistics provider such as, for example, a courierservice or a package handling service.

In some embodiments, the logistics interface 130 includes one or morecomputing devices such as, for example, a desktop computer, a cloudserver, a laptop computer, mobile devices, or any other computing deviceincluding hardware and software modules that execute on a processor andinteract with a memory. The logistics interface 130 may be connected,via wired or wireless connection via a network such as the Internet, tocomputing devices operated at, or associated with, the logisticsprovider.

The manufacturing portal 140 may be associated with the manufacturingfacility that manufactures the expanded cell therapy product. Themanufacturing portal 140 is operable to enable personnel at themanufacturing facility to control and record various processes duringthe manufacturing of the expanded cell product including, for example,maintaining a chain of identity and a chain of custody, recordinginformation relating to quality control assays performed during themanufacturing, recording transition between various processes, andproviding labels for containers of the cell therapy product during theexpansion process.

In some embodiments, the manufacturing portal 140 includes one or morecomputing devices such as, for example, a desktop computer, a cloudserver, a laptop computer, mobile devices, or any other computing deviceincluding hardware and software modules that execute on a processor andinteract with a memory. The manufacturing portal 140 may be connected,via wired or wireless connection, to computing devices operated at, orassociated with, the manufacturing facility.

In some embodiments, the manufacturing portal 140 includes a labelingmodule 142 and a COC/COI module 144. The labeling module 142 is operableto enable personnel at the manufacturing facility to generate labels forcontainers carrying the cell therapy product during the process ofmanufacturing the expanded cell therapy product (also referred to hereinas the expansion process). The labels may include information such as,for example, a patient-specific identifier, an identifier relating tothe personnel handling the container and/or performing the currentand/or previous step of the expansion process, results of a qualitycontrol assay performed at a previous step, a reason code relating tothe reason for generating the label, a barcode or a 2D code (e.g., a QRcode) identifying the cell therapy product with the patient-specificidentifier, and other suitable information.

The COC/COI module 144 is operable to enable users associated with themanufacturing facility to maintain an audit chain of custody during theexpansion process. The COC/COI module 144 is further operable to enableusers associated with manufacturing facility to maintain an audit chainof identity of the patient associated with the cell therapy productbeing expanded.

FIG. 3B illustrates the object schema for components of system 300 thatare suitably modified or built upon commercially available softwareplatforms in addition to those standard within those platforms. Forexample, a commercially available software platform may have built-inobjects corresponding to patient access (including, e.g., patientidentifier, patient contact information, patient authenticationinformation, etc.), treatment plan (including, e.g., an initial scheduleof patient treatment events), clinician access (e.g., clinicianidentifier, and clinician authentication information), and amanufacturing access (e.g., contact information for the manufacturingfacilities).

On the other hand, objects such as tumor procurement forms associatedwith the procedure for obtaining the solid tumor from the patient,manufacturing order including information relating to processes to beused for manufacturing the expanded cell therapy product and informationrelating to how the obtained solid tumor has been processed, schedule ofmanufacturing at one or more manufacturing facilities, label audit trailfor enabling audit of the entire process, may be custom-built tointerface with the built-in objects so as to provide the specificfunctionality associated with system 300.

The custom-built objects and the built-in objects are configured in thesystem 300 to interact with each other so as to enable maintaining andauditing COC and COI through all the events from obtaining the solidtumor from the patient to infusion of the expanded cell therapy productinto the patient. The custom-built objects, in concert with the built-inobjects, as well as schedule patient treatment events and associatedlogistics based on how the manufacturing processes progresses.

For example, in some embodiments, custom-built objects such as lotrecords and label audit trails may be provided in the system 300. Thesecustom-built objects, in addition to custom-built workflow automationand custom-built user profiles, provide the commercially availablesoftware platform with functionality such as, for example, maintenanceof COC/COI, audit capability, dynamic scheduling of patient treatmentevents based on manufacturing execution, label generation based onmanufacturing execution, and/or interaction between manufacturingexecution and procurement, that may not have been originally envisionedwithin the commercially available software platform. Additional detailsof the interaction between the custom-built objects and the built-inobjects for tumor procurement procedure, maintenance of COC and COI, andgeneration of labels during the manufacturing process are providedelsewhere herein.

B. Interaction Between Custom-Built and Built-In Objects for MaintainingCOC and COI

FIGS. 3C-3E schematically illustrate the tracking on biological materialthrough the manufacturing process at a manufacturing facility inaccordance with some embodiments of the present disclosure. In someembodiments, initially, when the tumor sample obtained from the patientat the clinical facility is delivered to the manufacturing facility, theshipping label is scanned and the information obtained from the scan isassociated with corresponding computer-based objects on the system. Theinformation may include, but is not limited to, cell order identifier,patient-specific identifier, time of tumor resection, processes used fortumor resection, processes used for storing the tumor followingresection, and expected processes to be used for expanding the celltherapy product from the tumor. Once patient-related informationobtained from the scan is verified with the cell order request that isseparately received at the manufacturing facility (e.g., via themanufacturing portal 140), the tumor is indicated in the system asreceived at the manufacturing facility, and the chain of custody passesto the manufacturing facility. Details about how the chain of custodyand chain of identity are maintained through the manufacturing processare described elsewhere herein.

The tumor is then moved for checking quality. During the quality check,the tumor-related information received from the scan is further verifiedto ensure that the tumor sample meets the requisite quality criteria.The information verified in this process includes, for example,information relating to the processes used for tumor resection and theprocesses used for storing and shipping the tumor following resection.The information relating to the processes used for tumor resection mayinclude, for example, various fields processed from a tumor procurementform which is further described in detail elsewhere herein. Once thetumor is deemed to meet the quality criteria, the corresponding label isscanned and the corresponding objects are updated. The updatedinformation included in the objects is accessible throughout the systemincluding, for example, at the hospital facility as well as thelogistics provider (also referred to herein as the courier facility). Insome embodiments, various processes associated with the hospitalfacility and the courier facility are updated based on the updatedobjects.

The tumor is then moved to manufacturing. A day zero batch record labelis then scanned to verify that the appropriate tumor sample is beingmoved manufacturing is to be processed by the appropriate manufacturingprocesses. The tumor is then moved to the appropriate flask (whichincludes the media and reagents for the expansion process by which thetumor for the patient is to be processed), and the label of the flask isscanned. The corresponding objects are updated using the informationobtained from the scans with appropriate time stamps.

The day zero flask is then manually moved to the incubator room fromwhere it is moved to the manufacturing room. In the manufacturing room,the flask label is again scanned and the information contained thereinrecorded. The information from the flask label is also used ensure thatthe appropriate manufacturing processes are followed.

After a first requisite amount of time (e.g., 11 days shown in FIG. 3C)based on the particular manufacturing process being followed, the flaskis seeded. During the seeding process, the flask may be required to beremoved from the manufacturing room. In such instances, the flask labelis scanned, and batch record is updated before and after the flaskleaves the manufacturing room. The information from the before and afterscans is matched before further processes is performed.

Following the seeding process, the flask is reintroduced into themanufacturing room. The flask label is scanned before and afterreintroducing into the manufacturing room following the seeding processand the batch records are updated. The information from the before andafter scans is matched to verify that the correct flask is being usedand the appropriate subsequent processes are used for further processingthe cell therapy product.

After a second requisite amount of time (e.g., at day 16 shown in FIG.3C), the cells from the seeded flask are split into a plurality of bags(e.g., 5 bags shown in FIG. 3C). Labels for each of the bags are scannedbefore and after reintroducing into the manufacturing room and the batchrecords and corresponding objects are appropriately updated. Theinformation from the before and after scans is used to verify that thecells from the flask are split in accordance with the cell orderrequest, and that the bags are further processed using the appropriatemanufacturing processes.

In some embodiments, a quality assay is performed at the first andsecond requite times to ensure that the manufacturing process isproceeding in accordance with the cell order request and following apredetermined schedule. In such embodiments, the results of the qualitycontrol assay may also be included in the scanned information. Uponobtaining such information from corresponding scans, if it is determinedthat the quality of the cell therapy product obtained at thecorresponding time point does not meet certain quality criteria, thebatch record and corresponding objects are updated accordingly. Ifnecessary, the schedule of the manufacturing process is also updated. Insome embodiments, the updated schedule may be communicated to thehospital facility as well as the courier so that the schedules forcorresponding processes to be performed at the hospital facility and bythe courier are suitable updated. The details of when and how theschedule of the manufacturing is updated are described elsewhere herein.

The manufacturing process is then continued (either as scheduled, orwith a suitably modified schedule and/or intermediate steps) in theplurality of bags to a predetermined end point. In some embodiments,cells from one of the plurality of bags are used for performing qualityassays on the final expanded cell therapy product. The bag selected fromthe quality assay as well as the bags containing cells to be released asthe final product are then labeled, and the labels scanned for updatingthe batch records and the corresponding objects.

The bags to be released as the final product are then input intocassettes and frozen for transportation to the hospital facility. Thelabels for cassettes are scanned and the batch records updated alongwith the corresponding objects. The label for the bag to be used forperforming quality control assays is then scanned, the batch record andthe corresponding objects updated, and the bag is moved to qualitycontrol station. Upon obtaining the results of the quality controlassay, the results are scanned and the batch records and correspondingobjects are updated.

In some embodiments, the bags to be released as the final product may bereleased to the courier (and ultimately to the hospital facility) with acaveat that the product in the bags has not yet been approved for use intherapy with the patient because the results of the quality assays arenot yet available. In such embodiments, the corresponding objects areupdated in real-time when the results of the quality assay are obtained,thereby reducing the time for delivery of the final cell therapy productto the patient.

Once the results of quality assay indicate that the final cell therapyproduct is approved for therapeutic use with the patient, the cassettesare scanned and the batch records and corresponding objects are updated.The cassettes are then prepared processed for transportation, handedover to the logistics provider (i.e., the courier), and the scanned,indicating that the chain of custody has passed to the courier.

The courier then transports the final cell therapy product to thehospital facility where the labels are scanned again to indicate thatthe chain of custody has passed to the hospital facility. The batchrecords and corresponding objects are updated so that the manufacturingfacility and the courier are notified that the final cell therapyproduct has been delivered to the hospital facility.

C. Labeling of Cell Therapy Product During Manufacturing Process andMaintenance of Chain of Custody and Chain of Identity

FIG. 3F schematically illustrates the process for maintaining COC andCOI through the journey of the cell therapy product from obtaining thesolid tumor through the manufacturing process to infusion into thepatient in accordance with some embodiments of the manufacturing process(e.g., a GEN 2 process, or a GEN 3 process). In some embodiments,initially, the hospital-side interface 110 receives information aboutthe patient from an employee (e.g., registered nurse) at a hospitalconnected with the hospital-side interface 110. Information about thepatient may include information about health insurance, personalidentifying information, health-related information, patient enrollmentinformation (e.g., consent forms) or any other information pertinent tothe patient that may be helpful in identifying and caring for thepatient.

After the employee, e.g., a clinician, of the hospital has determined apatient to be a candidate, for example, for a TIL infusion therapy, thepatient may provide consent to proceed with the TIL infusion therapythrough a computer connected to the hospital-side interface 110.

The employee of the hospital may then order TIL infusion therapy for thepatient via the events scheduler 120. A billing department employee ofthe hospital may then place a purchase order for the TIL infusiontherapy for the patient via the logistics interface 130. The TILinfusion therapy order and purchase order may be transmitted to thehospital-side interface 110. The hospital-side interface 110, mayconfirm receiving the order and purchase order as well as confirmingpatient enrollment and consent is complete before issuing a cell orderrequest to the events scheduler 120. Likewise, the hospital-sideinterface may communicate with the insurance provider to notify thatvarious patient treatment events have been scheduled and may alsoprovide the schedule of the patient treatment events to the insuranceprovider for processing the payment.

A patient-specific identifier may then be associated with the cell orderrequest and attached to the biological material associated with thepatient at every processing and/or shipping step to enable uninterruptedchain-of-custody and chain-of-identity tracking of the biologicalmaterial as detailed further herein. The patient-specific identifier mayalso be associated with the patient as well as the treatment equipmentused for administering the different patient treatment processes so asto keep track of the patient through the entire treatment regimen.

The patient-specific identifier may also be communicated to an insuranceprovider through the hospital-side interface 110 so as to enableprocessing of payment following various patient treatment events. Thepatient-specific identifier may be further communicated to the courierthrough the courier portal 130 to enable the courier to verify theidentity of the patient associated with the biological material beingtransported.

In various embodiments disclosed herein, any communication between thescheduling module 125 and the hospital-side interface 110, themanufacturing portal 140 or the logistics interface 130 relating to theschedule of manufacturing events and/or patient treatment events(whether changed or not) is accompanied with the patient-specificidentifier associated with the cell order request being processed andscheduled. Such communication including the patient-specific identifierenables the hospital-side interface 110, the manufacturing portal 140and the logistics interface 130 to accurately process, track andidentify the biological material, thereby avoiding misidentification ofthe biological material or patient, and improving patient safety.

Additionally, as explained in detail herein, the patient-specificidentifier is used for generating labels for the containers used duringthe expansion process for enabling maintenance of COC/COI through theexpansion process and enabling a post-facto audit for compliance withregulatory requirements.

In some embodiments, the patient-specific identifier may be included inthe cell order identifier structured to also be used for tracking achain of identity/chain of custody as well as for tracking eventhistory. In some embodiments, the cell order identifier, in addition tothe patient-specific identifier, may include several indicia or fields,each corresponding to the biological material from the patient havingundergone a processing step. FIG. 3G is a representative image of alabel for a cell therapy product in accordance with some embodiments. Insome embodiments, the label includes a cell order identifier 350, a 2Dcode (e.g., a QR code) 362, a reason field 370 and a product field 380.The cell order identifier 350 may include various fields such as, forexample, patient name 354, patient-specific identifier 352, patient'sdate of birth (e.g., for additional verification) 356, ahospital-associated patient identifier 358, and a manufacturing lotnumber 360.

Thus, the label enables maintenance of COI/COC and also enables trackingof materials from tumor resection at the clinical treatment center,shipment of tumor material to the manufacturing facility, manufacturingincluding testing of all in-process and finished product samples,shipment of drug product to the clinical treatment center, and infusionof the drug product.

In some embodiments, different labels may be generated at different timepoints during the journey of the cell therapy product from obtaining thesolid tumor through the manufacturing process to infusion into thepatient, such as those illustrated in FIG. 3H. The labels generated atdifferent time points may include additional or different informationthat that included in the label illustrated in FIG. 3G. For example, insome embodiments, the labels generated during the cell expansionprocess, that are e.g., affixed to the containers used during the cellexpansion process, may include a field corresponding to various timepoints and a result of a determination, made at the respective timepoint, of whether the acceptance parameters for the expansion celltherapy product meet certain acceptance criteria.

Thus, at any stage in the process, the label provides information aboutthe patient (via the patient-specific identifier) thereby enabling themaintenance of chain of identity and chain of custody. The label mayalso provide information about the various processing steps theassociated biological material has undergone, providing a record of thechain of custody as well as the event history, e.g., for audit purposes.

The system 300 may thus be configured to maintain COI and COC throughoutthe cell therapy product manufacturing process and supply chain.Additionally, the system 300 may be configured to include safeguards tomeet 21 CFR Part 11, HIPAA (Health Insurance Portability andAccountability Act), and GDPR (General Data Protection Regulation)regulations and to ensure that the data is secured and protected.

To limit and restrict access to sensitive data, the system 300implements users-based roles and access in some embodiments. In someembodiments, a time-stamp may be included on the label at the time ofits generation.

In some embodiments, the cell order identifier being tracked through thetracking module may further include indicia or fields associated with,e.g., various shipping/transportation events, various manufacturingprocess events, and/or various steps in the treatment regimen. Theseindicia or fields may not be printed on a label, but generated orappended by the tracking module for tracking of the biological materialassociated with the patient as well as for tracking the progress of thetherapy. In such embodiments, a time-stamp indicating a time ofcompletion of a certain step may be associated or appended with the cellorder identifier at every step where a given index or field is changedor updated. It will be appreciated that information included in suchupdated cell order identifier may also be used for dynamicallyrescheduling patient treatment events as discussed in detail elsewhereherein.

1. Labeling of Cell Therapy Product During Manufacturing Process

Referring back to FIG. 3F, in some embodiments, the cell orderidentifier is printed on readable labels and associated with barcodes orQR codes (or 2D codes), to allow adequate verification of identify fromtumor resection to infusion of the autologous drug product. The labelmay be affixed to objects associated with the biological material aswell as the treatment regimen. For example, the label including thepatient-specific identifier may be affixed to a container carrying theresected tumor from the hospital or clinical facility to the TILmanufacturing facility, the TIL manufacturing equipment and containersduring various steps of manufacturing, a shipping container for shippingthe expanded TILs back to the hospital or clinical facility, equipmentassociated with various treatment events, or any combination thereof.

For example, in some implementations, a patient may enter the system viathe patient access module 116 of the hospital-side interface 110 and isenrolled. Upon enrollment and capture within the system of the traceablepatient information, a patient-specific identifier (COI number) may beassigned to the patient by the system. The assignment of thepatient-specific identifier enables the scheduling of a resection dateand a manufacturing slot.

Scheduling of the manufacturing slot within the system triggersgeneration of a cell order and assignment of a lot number at themanufacturing site.

Prior to tumor resection, the clinical facility may access the system,confirm the correct patient identification, and generate a tumor bottlelabel and a tumor shipper label. The label may include thepatient-specific identifier in addition to the cell order identifier. Insome embodiments, the patient-specific identifier may be a field withinthe cell order identifier

Following tumor resection chain of custody is initiated at the trackingmodule 112. The labeled tumor bottle may then be placed in a shipper andhanded off to the courier for transport to the manufacturing site. Chainof custody continues as the courier verifies the cell order number onthe shipper label matches the cell order number on the shipping order inthe courier's system which is integrated into the system 300, therebyproviding real time tracking of the tumor between the treatment centerand the manufacturing site.

Upon delivery of the tumor to the manufacturing site and a proof ofdelivery may be generated within the system. Upon receipt of the tumor,manufacturing personnel enter the manufacturing lot number into systemat the manufacturing module 140 via the COC/COI module 144. Pulling upthe patient record (made available via the manufacturing coordinationmodule 118), the tumor bottle label is scanned into the system to verifythat the patient-specific identifier is associated with themanufacturing lot number. Upon verification, COC passes to themanufacturing site.

Personnel at the manufacturing facility may enter the system, whereinthe labeling module 142 enables generation of work in process (WIP)labels that contain the cell order request. In order to facilitate auditof records post-facto, the WIP labels may affixed to the batch recordand cell growth flask. FIG. 3G is a representative image of a WIP labelin accordance with an embodiment. FIG. 3H shows the different WIP typesof labels and the information included in each type of label inaccordance with an embodiment.

Manufacturing personnel may then scan the labels at key transitionpoints during the manufacturing process to verify the cell orderidentifier number on the cell growth flask matches the batch record,ensuring COI is maintained throughout the manufacturing process.

Additionally, each Quality Control (QC) sample will have its own labelwith the COI number to assure that the results generated are then linkedto the corresponding batch record. Upon completion of the manufacturingprocess, the labeling module 142 may generate finished product labels.FIGS. 3I and 3J are examples of a finished product label.

As seen in FIGS. 3I-3J, these finished product labels contain uniquepatient identification including name 354, date of birth (DOB) 356, andthe patient-specific identifier 352. Finished product labels are affixedto the final product and both batch record and labels are scanned toensure COI and complete chain of custody within the manufacturingfacility.

The finished product is placed in the shipper with the same cell orderidentifier including a matching patient-specific identifier, verifiedand transferred to the courier for delivery to the treatment center forinfusion. The courier may then verifies that the cell order identifierand the patient-specific identifier on the shipping container matchesthe cell order and patient information in the courier's system receivedthrough the logistics interface 130, thereby providing near real timetracking of each drug product lot between the manufacturing site and thetreatment center. At this time, COC is transferred from themanufacturing site to the courier.

The courier delivers the finished product to the treatment center forinfusion into the patient. The courier may initiate proof of delivery(POD) through the logistics interface 130, and COC is passed from thecourier to the treatment center. COI ends with infusion of the productto the patient at the treatment center.

As discussed herein, communicating the patient-specific identifierbetween the hospital-side interface 110, the events scheduler 120, themanufacturing portal 140 and the logistics interface 130 after eachprocessing step ensures that all involved parties (e.g., the clinicalfacility, the manufacturing facility and the logistics provider) receivea record of the chain of custody as well as chain of identify of thebiological material, and also receive a record of the various processsteps the biological material has undergone.

The patient-specific identifier and the cell order request provided tothe solid tumor via a label thus, enable tracking of the shipment so asto maintain chain of custody and chain of identification that isnecessary for patient safety as well as for regulatory compliance (e.g.,FDA audits).

2. Label Reconciliation in Case of Changes to Manufacturing Process

Once the solid tumor is received at the manufacturing facility,manufacturing of the cell therapy product is initiated in accordancewith the cell order request. For example, at least a portion of thesolid tumor may be used for manufacturing the cell therapy product usinga cell expansion technique.

In some embodiments, a computer subsystem associated with themanufacturing facility, e.g., the labeling module 142, may cause asecond label to be printed to be associated with the portion of thesolid tumor being used for cell expansion. The second label may includethe patient-specific identifier and the cell request identifier. Thesecond label further enables the tracking of the cell therapy productfor maintaining the chain of custody and chain of identification.

Subsequently, during the manufacturing of the cell therapy productacceptance parameters of for the manufactured cell therapy product atvarious time points are determined. The acceptance parameters are thencompared, at the acceptance determining module 123, to determine whetherthe acceptance parameters determined at a particular time point meetacceptance criteria at the corresponding time point. The acceptanceparameters may be determined by any suitable method or process disclosedherein. Additionally, the acceptance criteria may be any acceptancecriteria disclosed herein.

In some embodiments, the result of the determination of the acceptanceparameters and whether the acceptance parameters meet the acceptancecriteria may be appended to the cell order identifier as disclosedelsewhere herein. In some embodiments, a third label may be generatedbased on the result of the determination of whether the acceptanceparameters meet (or do not meet) the acceptance criteria.

For example, if the acceptance parameters at a second time point do notmeet the acceptance criteria at the second time point, the third labelis generated for the container used during the manufacturing process toinclude a “reason code” to convey the information that the acceptanceparameters at the second time point do not meet the acceptance criteriaat the second time point and why the acceptance criteria at the secondtime point are not met.

Further, if it is determined that the cell therapy product obtained atthe second time point may be reprocessed from a first time pointpreceding the second time point to appropriately obtain a cell therapyproduct that meets the acceptance criteria at the second time point, thethird label may include information such as, for example, a new orupdated cell request identifier as disclosed elsewhere herein. In suchinstances, the third label may additionally include a “reason code” toconvey the information relating to why the acceptance criteria at thesecond time point and why it was appropriate to reprocess the celltherapy product from the first time point.

Next, based on whether the acceptance criteria at one or more timepoints are met, as determined at the acceptance determination module123, the manufacturing schedule for manufacturing of the cell therapyproduct is suitably modified to generate an updated manufacturingschedule as discussed elsewhere herein. In addition, the availability ofmanufacturing slots at the manufacturing facility is also suitablymodified to reflect the changes, if any, in the current schedule ofmanufacturing of the cell therapy product. The changed or updatedavailability of manufacturing slots at that manufacturing facility maythen be communicated to the computing device. Further, the preliminaryschedule of patient treatment events may also be modified based onupdated manufacturing schedule, and an updated schedule of patienttreatment events may be communicated to the computing device.

The manufacturing of the cell therapy product is then completed inaccordance with the updated manufacturing schedule.

In some embodiments, a manufacturing label corresponding to each of theplurality of time points at which the acceptance parameters aredetermined is generated. The manufacturing label at each time point mayinclude updated information associated with quality of manufactured celltherapy product. The updated information may include the acceptanceparameters at the corresponding time point and/or whether the acceptanceparameters meet the acceptance criteria at that time point.

In addition, in some embodiments, a controller controlling themanufacturing process may be configured to read an updated manufacturinglabel and determine the subsequent processing step. For example, if anupdated manufacturing label following QA test at a second time pointindicates that the cell therapy product does not meet certain acceptancecriteria, but the cell therapy product may be reprocessed from the firsttime point, the controller may cause suitable changes in themanufacturing process. In addition, suitable changes to themanufacturing schedule, the availability of manufacturing slots and theschedule of patient treatment events are also made based on theinformation on the updated label. Those of skill in the art willappreciate that the updated manufacturing label may include the cellorder identifier and the patient-specific identifier in addition to theinformation relating to the quality of the manufactured cell therapyproduct and the reason code to facilitate chain of identification andchain of custody.

Those of skill in the art will further appreciate that the informationrelating to the changes to the manufacturing schedule, the availabilityof manufacturing slots and the schedule of patient treatment events, asobtained from reading the updated manufacturing labels may becommunicated to the hospital-side interface and the logistics interfaceto enable corresponding computing devices to make corresponding changesto respective schedules associated with those entities.

D. Coordinating Manufacturing of Cell Therapy Product with PatientTreatment Events

FIGS. 4A and 4B show a flow chart for a method for coordinating themanufacturing of TILs for a patient in accordance with an embodiment ofthe present disclosure. In some embodiments, the method illustrated inFIGS. 4A and 4B is implemented on a system as a whole depicted in FIG.3A, while in some embodiments, the method illustrated in FIGS. 4A and 4Bis implemented by portions of the system depicted in FIG. 3A. In someembodiments, the events scheduler 120 receives, e.g., at S305, the cellorder request to expand the cell therapy product, e.g., T-cells, for thepatient from the hospital-side interface 110, and the patient-specificidentifier associated with the cell order request.

In some embodiments, the patient-specific identifier or alternately acell order identifier is generated by the events scheduler 120, e.g., atS310, rather than the tracking module 112.

The cell order request may include information such as, for example,patient identifying information, target dates for various patienttreatment events, and/or target parameters for the final expanded TILmanufacturing product. Once the cell order request is received, theevents scheduler 120 may transmit a confirmation to the hospital-sideinterface 110 indicating that the cell order request corresponding tothe patient-specific identifier has been received. In embodiments wherethe patient-specific identifier is generated by the events scheduler120, the events scheduler 120 may transmit the confirmation indicatingthat the cell order request for the patient has been received andprovide the patient-specific identifier or the cell order identifier tothe hospital-side interface 110. In addition, the events scheduler 120may transmit a target schedule of patient treatment events based on thereceived cell order request.

In addition, the events scheduler 120 may transmit an initiation requestincluding the patient specific identifier, e.g., at S315, to thehospital-side interface 110, e.g., to a clinical facility, to perform aprocedure on the patient to obtain a solid tumor from the patient andschedule a courier pick-up time to transfer the obtained solid tumor tothe manufacturing facility. As discussed herein, the procedure mayinclude, but is not limited to, tumor biopsy for extracting a portion ofa tumor from the patient. In some embodiments, the events scheduler 120may receive a procedure date from the employee using the hospital-sideinterface 110. In response, the events scheduler 120 may prompt theemployee to order supplies (e.g., tumor resection kit, tumor shippingcontainer or cryo-shipping container) as needed and associate them withthe patient-specific identifier.

After the procedure to obtain the solid tumor is performed and the solidtumor is shipped, e.g., at S320, and once the obtained solid tumor arereceived at the manufacturing facility, the scheduling module 125dynamically schedules, e.g., at S325, various manufacturing events aswell as various patient treatment events and associates each event withthe patient-specific identifier. In some embodiments, the schedulingmodule 125 determines the schedule for manufacturing events and patienttreatment events before receiving the obtained solid tumor such as, forexample, upon receiving information about a scheduling of the procedureto obtain the solid tumor from the patient from the hospital-sideinterface 110, and associates each event with the patient-specificidentifier.

The schedule for various manufacturing events is associated with thepatient-specific identifier and may include corresponding target datesat which various manufacturing steps (e.g., steps A-F shown in FIG. 2A,2B or 2C and/or FIG. 9 ) are performed as well as a corresponding targetdate for the completion of the TIL manufacturing process. As discussedherein, the target dates for various manufacturing steps may depend onfactors such as the size of the tumor received, cell counts beforeinitiating a given step, numerical folds at the end of a given step,number of days needed to achieve a certain numerical fold during a givenstep, and/or other factors.

The schedule for the various patient treatment events may include targetdates for various patient treatment events associated with thepatient-specific identifier such as, for example, target TIL infusiondate, a lymphodepletion date, an inpatient stay duration, a schedule forIL-2 infusion regimen, and/or other similar treatment related dates. Asdiscussed herein, the schedule for the patient treatment events isdependent on the schedule for the manufacturing events, and inparticular on the TIL manufacturing completion date. The schedule forthe various patient treatment events may be transmitted to thehospital-side interface 110 along with the patient-specific identifierin some embodiments.

The scheduling module 125 is configured to dynamically change theschedule for manufacturing events as well as the schedule for patienttreatment events based on the progress and success of differentmanufacturing steps. For example, depending on whether the acceptanceparameters associated with the expansion cell therapy product atdifferent steps meet certain acceptance criteria, the dates at whichsubsequent manufacturing steps are initiated need to be changed, which,in turn, changes the target completion date, and as a consequence, thedates for the patient treatment events need to be changed using, forexample, the methods depicted in FIG. 4B or 4C.

The acceptance determining module 123 determines whether the acceptanceparameters associated with the expansion cell therapy product meetcertain acceptance criteria. The acceptance parameters include, but arenot limited to, cell count, cell viability, sterility, mycoplasma count,CD3 count, CD5 count, interferon γ (INF-γ) production, result of anendotoxin assay, result of a Gram stain assay, etc. Some or all of theseacceptance parameters may need to meet acceptance criteria depending onthe step at which the acceptance parameters are measured. For example,at the end of first priming expansion (also referred to herein as thefirst time point), e.g., at 11 days from initiation of the expansion,the acceptance criteria may be that the cell count should be at least5×10⁶ viable cells. The acceptance criteria at different steps alsodepend on the process (e.g., Gen 2, Gen 3, Gen 3.1, etc.) being used toperform the expansion.

Thus, the acceptance determining module 123 may determine whether theacceptance parameters meet certain acceptance criteria at more than onedifferent time points following the initiation of expansion depending onthe process being used to perform the expansion. In other words, theacceptance determining module 123 performs a quality test, also referredto herein as a QA test, at each of different specified time points todetermine the next course of action, and the scheduling module 125dynamically schedules subsequent manufacturing steps and patienttreatment events based on the results of the QA tests obtained from theacceptance determining module 123.

One example of acceptance parameters and acceptance criteria for finalproduct testing for Gen 2, Gen 2 like and Gen 3 process is provided inTable B.

TABLE B Test Type (Acceptance Parameter) Method Acceptance CriteriaRelease Testing Cell viability Fluorescence ≥70% Total ViableFluorescence 1e9 to 150e9 Cell Count Identity Flow Cytometry Gen 2-like:(% CD45+/CD3+) ≥90% CD45+CD3+ TIL for all Indications Gen 3: ≥90%CD45+CD3+ TIL for Non-Ovarian ≥85% CD45+CD3+ TIL for OvarianInterferon-gamma Stimulation and ≥500 pg/mL production ELISA (Stimulated− Unstimulated)

Referring to FIG. 4B, in an embodiment, following the initiation ofexpansion of the cell therapy product, the acceptance determining module123 determines, e.g., at S402, whether the expanded cell therapy productat first time point pass a QA test associated with the first time point(e.g., following Step B shown in FIG. 2A and/or FIG. 9 ). Upon adetermination that the cells do not pass the QA test at the first timepoint, e.g., at S404, the acceptance determining module 123 determineswhether the cells did not pass the QA test because of contamination. Ifthe cells did not pass the QA test because of contamination, the case isreviewed for potential termination, e.g., at S422.

On the other hand, if the cells did not pass the QA test because ofreasons other than contamination, e.g., because of low cell count or lowviability, the scheduling module 125 may extend the ongoing step for atime depending on the reasons due to which the cells did not pass the QAtest, and review the results from the final harvest, e.g., at S408. Insome embodiments, all subsequent manufacturing steps and patienttreatment events may be rescheduled as a consequence of extending theongoing step for a time.

Alternately, the scheduling module 125 may reschedule only the patienttreatment events so as to account for a potential delay in completion ofthe manufacturing process or to account for a potential cryogenicfreezing of the manufactured cell therapy product to allow for a timelag between completion of manufacturing and infusion of the cell therapyproduct. It will be appreciated that in some embodiments, the celltherapy product comprises T-cells of the patient. In some embodiments,the cell therapy product comprises tumor infiltrating lymphocytes(TILs). Thus, the discussion herein may, for the sake of simplicity,refer to the cell therapy product interchangeably as T-cells or TILs.

For example, in some cases, the cell therapy product may pass a final QAtest despite not passing the QA test at the first time point. In suchcases, it may be prudent to deviate from an ideal schedule formanufacturing events and patient treatment events, and delaylymphodepletion until the final QA test is performed so as to avoidunnecessary patient treatment. Thus, depending on the reasons for whichthe cells did not pass the QA test at the first time point, it isdetermined, at S418, whether the schedule of the patient treatmentevents needs to deviate from the ideal schedule for the patienttreatment events. Upon a determination that the schedule of patienttreatment events needs to deviate from the ideal schedule, thescheduling module 125 may reschedule (i.e., delay), e.g., at S420,lymphodepletion as well as subsequent patient treatment events, andfurther optionally schedule a cryogenic freezing step following thecompletion of manufacturing process.

Referring back to S402, if the cells pass the QA test at the first timepoint, no changes may be needed to the schedule of the manufacturingprocess or the patient treatment events. The acceptance determiningmodule 123 then determines, e.g., at S406, whether the expanded T-cellsat a second time point pass a QA test associated with the second timepoint (e.g., following Step C or Step D shown in FIG. 2A and/or FIG. 9).

If the cells do not pass the QA test at the second time point, theacceptance determining module 123 determines, e.g., at S404, whether thecells did not pass the QA test because of contamination. If the cellsdid not pass the QA test because of contamination, the case is reviewedfor potential termination, e.g., at S422.

On the other hand, if the cells did not pass the QA test because ofreasons other than contamination, e.g., because of low cell count or lowviability, the scheduling module 125 may extend the ongoing step for atime depending on the reasons due to which the cells did not pass the QAtest, and review the results from the final harvest, e.g., at S408. Insome embodiments, all subsequent manufacturing steps and patienttreatment events may be rescheduled as a consequence of extending theongoing step for a time.

Alternately, the scheduling module 125 reschedule only the patienttreatment events so as to account for a potential delay in completion ofthe manufacturing process or to account for a potential cryogenicfreezing of the manufactured TILs to allow for a time lag betweencompletion of manufacturing and infusion of the TILs.

For example, in some cases, the cells may pass a final QA test despitenot passing the QA test at the second time point. In such cases, it maybe prudent to deviate from an ideal pre-decided schedule (also referredto herein as of the golden path) of patient treatment events and delaylymphodepletion until the final QA test is performed so as to avoidunnecessary patient treatment. Thus, depending on the reasons for whichthe cells did not pass the QA test at the second time point, thescheduling module 125 may reschedule (i.e., delay), e.g., at S420,lymphodepletion as well as subsequent patient treatment events, andfurther schedule a cryogenic freezing step following the completion ofmanufacturing process.

In some embodiments, when the cells fail the QA test at the first orsecond time point, the patient-specific identifier may be updated toidentify that the cells have failed the QA test at the respective timepoint, and the hospital-side interface 110 and the logistic interface130 are notified that there is a possibility that the patient treatmentevents for the patient (associated with the patient-specific identifier)may have to be canceled or delayed. Where the schedule of patienttreatment events has deviated from the pre-decided schedule (e.g., thegolden path schedule) and the patient treatment events have beenrescheduled, the hospital-side interface 110 and the logistics interface130 are notified that the schedule corresponding to the patient-specificidentifier has been updated, and the updated schedule is communicated tothe hospital-side interface 110 and the logistics interface 130.

Such notification enables the clinical facility or the hospital tosuitably adjust the various schedules for the patient treatment eventsas well as make necessary logistical arrangements relating toavailability of appropriate personnel and availability of facilities andequipment needed for the respective patient treatment events. Thenotification may additionally facilitate the hospital or the clinicalfacility to inform the patient of the change in the schedule of thepatient treatment events. Alternately, the events scheduler 120 and/orthe hospital-side interface 110 may communicate with the patientdirectly to notify the patient of the change in the schedule of patienttreatment events and enable the patient to coordinate with the hospitalor the clinical facility for arranging the patient treatment eventsaccording to the changed schedule.

In some embodiments, the hospital-side interface 110 may additionallycommunicate the updated schedule to the insurance provider so thatappropriate measures for processing the payment may be taken.

Similarly, the notification enables the logistics provider(s) to makenecessary arrangements for rescheduling the shipment of the biologicalmaterial such as, for example, arrangement of appropriate shipping boxesand availability of appropriate personnel for handling the biologicalmaterial.

Referring back to S406, if the cells pass the QA test at the second timepoint, the scheduling module 125 may maintain the schedule for thepatient treatment events. For example, in some embodiments, thescheduling module 125 may communicate, e.g., at S410, to thehospital-side interface 110 that a lymphodepletion treatment may beadministered to the patient based on the pre-decided schedule with acaveat that there is a low but non-zero probability that themanufacturing process may yet be delayed or may not yield the desiredfinal product. Such communication is designated in FIG. 4B as“lymphodepletion at risk.” No changes are made to the schedule of themanufacturing process or the patient treatment events in such asituation.

The acceptance determining module 123 then determines, e.g., at S412,whether the expanded T-cells at a third time point pass a QA testassociated with the third time point (e.g., following Step D or Step Eshown in FIG. 2A and/or FIG. 9 ).

If the cells pass the QA test for the third time point, the expandedcells are deemed to be ready for product release at the target TILinfusion date. The scheduling module 125, in such cases, notifies, e.g.,at S424, the hospital-side interface 110 that the patient treatmentevents are to continue at the target schedule. In other words, no changeis made to the schedule.

On the other hand, if the cells do not pass the QA test for the thirdtime point, a product impact assessment is performed (e.g., by thedoctor or the chief medical officer administering the treatment), e.g.,at S414, to determine whether the expanded cells can be provided forinfusion or the treatment terminated depending on the reasons for whichthe cells did not pass the QA test for the third time point. If, uponthe product impact assessment, it is determined, e.g., at S416, that thetreatment can move forward with the caveat that there may be certainrisk associated with continuing the treatment with the availableproduct, the expanded cells are approved for release, e.g., at S424,without any change in the schedule of the patient treatment events.

Referring to FIG. 4C, in an embodiment, following the initiation ofexpansion of the cell therapy product, the acceptance determining module123 determines, e.g., at S402, whether the expanded cell therapy productat first time point passes a QA test associated with the first timepoint (e.g., following Step B shown in FIG. 2A and/or FIG. 9 ). Upon adetermination that the cells do not pass the QA test at the first timepoint, e.g., at S404, the acceptance determining module 123 determineswhether the cells did not pass the QA test because of contamination. Ifthe cells did not pass the QA test because of contamination, the case isreviewed for potential termination, e.g., at S422.

On the other hand, if the cells did not pass the QA test because ofreasons other than contamination, e.g., because of low cell count or lowviability, the scheduling module 125 may extend the ongoing step for atime depending on the reasons due to which the cells did not pass the QAtest, and review the results from the final harvest, e.g., at S408. Insome embodiments, all subsequent manufacturing steps and patienttreatment events may be rescheduled as a consequence of extending theongoing step for a time.

Referring back to S402, if the cells pass the QA test at the first timepoint, no changes are made to the schedule of the manufacturing processor the patient treatment events. The acceptance determining module 123then determines, e.g., at S406, whether the expanded T-cells at a secondtime point pass a QA test associated with the second time point (e.g.,following Step C or Step D shown in FIG. 2A and/or FIG. 9 ).

If the cells do not pass the QA test a the second time point, theacceptance determining module 123 determines, e.g., at S404′, whetherthe cells did not pass the QA test because of contamination. If thecells did not pass the QA test because of contamination, the case isreviewed for potential termination, e.g., at S422.

On the other hand, if the cells did not pass the QA test because ofreasons other than contamination, e.g., because of low cell count or lowviability, the acceptance determining module 123 determines, e.g., atS458, whether the cell therapy product obtained at the second time pointis of sufficient quality to enable re-performing of the cellmanufacturing process from first time point so as to result in the celltherapy product at the second time point with acceptance parameters thatmeet the acceptance criteria at the second time point. Suchdetermination may be made based on the acceptance parameters of the celltherapy product obtained at the second time point. As an example, if theacceptance parameters at the second time point meet the all acceptancecriteria at the second time point except the total viable cell count, itmay be viable to re-perform the cell manufacturing process between thefirst and second time points to obtain the requisite viable cell count.

If it is determined that re-performing of the cell manufacturing processfrom the first time point is not viable, the case is reviewed forpotential termination, e.g., at S422.

On the other hand, if it is determined that re-performing of the cellmanufacturing process between the first and second time points isviable, the scheduling module 125 estimates a time of completion of thecell manufacturing process following the re-performing of the processbetween the first and second time points, e.g., at S460. It will beappreciated that the time needed to re-perform the cell manufacturingprocess between the first and second time points may be estimated basedon the acceptance parameters of the cell therapy product obtained at thesecond time point. Thus, the time needed to complete the cell expansionprocess, including the re-performing of the process from the first timepoint, for manufacturing the cell therapy product may be dependent onthe acceptance parameters of the cell therapy product obtained at thesecond time point.

The scheduling module 125 may then reschedule all subsequentmanufacturing steps and patient treatment events as a consequence ofre-performing the cell manufacturing process from the first time pointbased on the acceptance parameters of the cell therapy product obtainedat the second time point. For example, the scheduling module 125 mayreschedule the patient treatment events so as to account for the delayin completion of the manufacturing process or to account for a potentialcryogenic freezing of the manufactured TILs to allow for a time lagbetween completion of manufacturing and infusion of the TILs.

On the other hand, if the acceptance parameters of the cell therapyproduct obtained at the second time point meet all the acceptancecriteria, the cell manufacturing process may be continued as originallyscheduled.

Those of skill in the art will appreciate that even after re-performingof the cell manufacturing process from the first time point upondetermination that such re-performing is viable, the acceptanceparameters of the cell therapy product obtained at the repeated secondtime point (also referred to herein as alternate second time point) aredetermined. It is then again determined if the acceptance parameters atthe repeated second time point (which may be different from the originalsecond time point in the cell manufacturing process) meet the acceptancecriteria for the second time point before continuing the cellmanufacturing process past the second time point.

The continued process then follows the same path “B” as that shown inFIG. 4B in some embodiments. For example, in some cases, the cells maypass a final QA test despite not passing the QA test at the second timepoint. In such cases, it may be prudent to deviate from an idealpre-decided schedule (also referred to herein as of the golden path) ofpatient treatment events and delay lymphodepletion until the final QAtest is performed so as to avoid unnecessary patient treatment. Thus,depending on the reasons for which the cells did not pass the QA test atthe second time point, the scheduling module 125 may reschedule (i.e.,delay), e.g., at S420, lymphodepletion as well as subsequent patienttreatment events, and further schedule a cryogenic freezing stepfollowing the completion of manufacturing process.

Those of skill in the art will appreciate that when a cell manufacturingprocess has several time points for determining acceptance parametersand whether those acceptance parameters meet the acceptance criteria atthose corresponding time points, it may be viable to re-perform thesteps in the cell manufacturing process from an immediately prior timepoint. Thus, the terms “first time point” and “second time point” do notnecessarily describe numerically first and numerically second timepoints at which acceptance parameters are determined. Instead, the“first time point” refers to a time point in the cell manufacturingprocess from which it may be viable to re-perform the steps of the cellmanufacturing process if the acceptance parameters at a subsequent timepoint do not meet the acceptance criteria at that time point. Forexample, in a 1C-Process (see, FIG. 6 ), the first and second timepoints may be Day 27 and Day 30 respectively, Day 30 and Day 36respectively, or Day 36 and Day 43 respectively. Similarly, in a2A-Process (see, FIG. 6 ), the first and second time points may be Day11 and Day 16 respectively, or Day 16 and Day 22 respectively.

In some embodiments, when the cells fail the QA test at the first orsecond time point, the patient-specific identifier may be updated toidentify that the cells have failed the QA test at the respective timepoint, and the hospital-side interface 110 and the logistic interface130 are notified that there is a possibility that the patient treatmentevents for the patient (associated with the patient-specific identifier)may have to be canceled or delayed.

Because the schedule of manufacturing of the cell therapy product maydeviate from the ideal schedule if the re-performing of the cellmanufacturing process at the first time point is viable, schedule ofpatient treatment events also deviates from the pre-decided schedule(e.g., the golden path schedule). Accordingly, the scheduling modulereschedules the patient treatment events, and the hospital-sideinterface 110 and the logistics interface 130 are notified that theschedule corresponding to the patient-specific identifier has beenupdated. The updated schedule is communicated to the hospital-sideinterface 110 and the logistics interface 130.

As discussed herein, such notification about the change in scheduleenables the clinical facility or the hospital to suitably adjust thevarious schedules for the patient treatment events. Additionally,necessary logistical arrangements relating to availability ofappropriate personnel and availability of facilities and equipmentneeded for the respective patient treatment events can be made. Thenotification may further facilitate the hospital or the clinicalfacility to inform the patient of the change in the schedule of thepatient treatment events. Alternately, the events scheduler 120 and/orthe hospital-side interface 110 may communicate with the patientdirectly to notify the patient of the change in the schedule of patienttreatment events and enable the patient to coordinate with the hospitalor the clinical facility for arranging the patient treatment eventsaccording to the changed schedule.

In some embodiments, the hospital-side interface 110 may additionallycommunicate the updated schedule to the insurance provider so thatappropriate measures for processing the payment may be taken.

Similarly, the notification enables the logistics provider(s) to makenecessary arrangements for rescheduling the shipment of the biologicalmaterial such as, for example, arrangement of appropriate shipping boxesand availability of appropriate personnel for handling the biologicalmaterial.

In some embodiments, unless the treatment is terminated, the schedulingmodule 125 communicates with the logistics interface 130 to provide apick-up order based on the completion date determined based on theresults of the QA test at various time points. The logistics interface130 then communicates with a logistics provider (not shown) to makesuitable arrangements for timely collecting and shipping the expandedTILs to the hospital or clinical facility for subsequent patienttreatment (e.g., infusion). For example, in some embodiments, thelogistics provider may be required to ship a specialized container forhandling a biological sample to the hospital or clinical facility acertain number of days before a scheduled patient treatment event.

Similarly, if it is determined that the expanded cells pass the QA test,the scheduling module 125 communicates the scheduled completion date andthe schedule of patient treatment events (i.e., an updated schedule) tothe hospital-side interface 110. On the other hand, if it is determinedthat the treatment is to be terminated, the scheduling module 125communicates with the hospital-side interface 110 that the treatment isto be terminated.

In some embodiments, upon rescheduling the patient treatment event, anupdated schedule for the patient treatment events is transmitted to thehospital-side interface 110 along with the associated patient-specificidentifier.

In some embodiments, association between the cell order identifier andthe patient-specific identifier may be updated if it is determined thatre-performing the steps of the cell manufacturing process from the firsttime point viable. For example, upon determination that re-performing ofthe expansion of the cell therapy product from the first time point isviable, the cell order identifier is dissociated from thepatient-specific identifier. A new cell order identifier associated withthe cell order request may be generated and the patient-specificidentifier is associated with the new cell order identifier. The newcell order identifier may be generated based on the acceptanceparameters determined at the second time point, in some embodiments. Thecell order identifier, in some embodiments, may include fieldscorresponding to each time point at which the acceptance parameters forthe cell therapy product are determined. In addition, in someembodiments, the cell order identifier may also include the result ofthe determination of whether the acceptance parameters meet theacceptance criteria, including, for example, which acceptance parametersmeet the acceptance criteria and the value of the acceptance parameters.

In some embodiments, the patient-specific identifier, the new cell orderidentifier and an estimated time of completion of the expansion of thecell therapy product is transmitted to the hospital-side interface toenable the hospital-side interface to track the cell therapy product andassociate the cell therapy product with the patient.

The various fields included in the cell order identifier may enable thescheduling module 125 to determine a new schedule for the patienttreatment events as well as the shipping and logistics events associatedwith the patient treatment events based on the updated or new cell orderidentifier generated at various time points. The new schedule is thentransmitted to the logistics interface along with the associatedpatient-specific identifier. The patient-specific identifier and theupdated or new cell order identifier may also be transmitted to thelogistics interface along with the new schedule in some embodiments soas to maintain chain of custody and chain of identification for the celltherapy product.

In some embodiments, if it is determined that re-performing of the stepsof the cell manufacturing process is not viable, e.g., because theacceptance parameters at the second time point do not meet a certainthreshold for the acceptance criteria, the patient treatments subsequentto the second time point may be canceled. In such embodiments, thecancellation of the patient treatments is communicated to thehospital-side interface and the logistics interface. In someembodiments, the expanded cell therapy product may be destroyed upondetermining that re-performing of the steps of the cell manufacturingprocess is not viable. For example, in instances where obtaining aviable expanded cell therapy product may not be possible if the steps ofthe cell manufacturing process cannot be re-performed, the infusion ofthe cell therapy product becomes impossible. Thus, it may be detrimentalto the patient (e.g., either in terms of health outcomes, or in terms offinancial outcomes or both) to continue patient treatment eventssubsequent to the second time point. Additionally, if it is determinedbased on the determined acceptance parameters at the second time thatthe expanded cell therapy product at the second time point cannot befurther used (e.g., if the cell therapy product at the second time pointis contaminated or does not meet certain other acceptance criteria), thecell therapy product may be destroyed. In such instances, the cell orderidentifier is dissociated from patient-specific identifier.

E. Coordinating Manufacturing Slots Between Manufacturing Facilities

In a further aspect of the present disclosure, a method of manufacturinga cell therapy product for a patient is disclosed. In some embodiments,the method includes receiving a cell order request to manufacture thecell therapy product for the patient. The cell order request may bereceived at a computing device associated with a clinical facility. Thecell order request may be received via, e.g., a hospital-side interface110. In some embodiments, the computing device may generatepatient-specific identifier and a cell order identifier upon receivingthe cell order request, and associate the patient-specific identifierand the cell order identifier with the cell order request.

In addition to the cell order request, the computing device may receivemanufacturing slots at a plurality of manufacturing facilities formanufacturing the cell therapy product. For example, the computingdevice may be communicatively coupled to computer subsystems associatedwith the plurality of manufacturing facilities via, e.g., the Internet,such that the computing device may receive information relating to themanufacturing slots in response to a query by the computing device. Themanufacturing slots for a respective manufacturing facility may beindicative of the availability of equipment and personnel at thatmanufacturing facility to enable that manufacturing facility tomanufacturing the cell therapy product in accordance with the cell orderrequest.

The computing device may further receive a preliminary schedule ofpatient treatment events for treating the patient with the cell therapyproduct. The preliminary schedule may be determined based on an idealschedule of manufacturing the cell therapy product assuming that thecell therapy product meets the QA criteria at each manufacturing stepduring the manufacturing process as discussed elsewhere herein.

Upon receiving the cell order request, the manufacturing slots, and thepreliminary schedule of patient treatment events, the computing devicemay determine and display, in a scheduling user interface, a pluralityof available manufacturing slots for manufacturing the cell therapyproduct based on the preliminary schedule of patient treatment events.The available manufacturing slots are determined based on factors suchas, for example, the location of the clinical facility, the location ofthe respective manufacturing facilities, the availability of logisticsproviders for shipping the cell therapy product between the respectivemanufacturing facility and the clinical facility, the availability ofclinical personnel at the clinical facility so as to perform thenecessary treatment procedures associated with the patient treatmentevents, and any other factors that may affect the timing of the arrivaland/or use of the cell therapy product at a respective location.

One of the available manufacturing slots is then selected either by anoperator of the computing device or automatically by the computingdevice. Upon selection of an available manufacturing slot, a solid tumormay be obtained from the patient, e.g., at the medical facility, byperforming an appropriate procedure. The procedure may be performed inaccordance with the preliminary schedule and based on the availablemanufacturing slot in consideration of the timing of shipping andavailability of logistics provider the solid tumor to the respectivemanufacturing facility. In some embodiments, the computing device mayinitiate printing of a first shipper label (see e.g., row 1 of FIG. 3H)to be associated with the solid tumor. The first shipper label mayinclude the patient-specific identifier and the cell order identifier.In some embodiments, the first label may additionally includeinformation relating to the clinical facility, the selectedmanufacturing facility and the logistics provider. Additionally, a firstproduct label (see, e.g., row 2 of FIG. 3H) to be associated with theproduct container. An example of the first product label is illustratedin FIG. 3G.

The solid tumor is then transferred to the selected manufacturingfacility in accordance with the available manufacturing slot. Those ofskill in the art will appreciate that because the solid tumor includeslive cells, the timing of arrival of the solid tumor at the selectedmanufacturing facility should be carefully coordinated with theavailability of the manufacturing slot so that the manufacturing stepsmay be initiated with an acceptable time window. Thus, the transfer ofthe solid tumor from the clinical facility to the selected manufacturingshould be carefully coordinated based on the availability of thelogistic provider, as well as other factors that may affect suchtransfer. For example, there may be weather-related or traffic delaysthat can be anticipated in some instances and thus, the transferschedule may be adjusted accordingly. In other instances, the delays intransfer may not be foreseeable. However, even in such instances, thetransfer of the solid tumor from the clinical facility to the selectedmanufacturing facility may be coordinated in other suitable ways.

F. Tumor Procurement Protocol

Referring back to FIG. 3A, the hospital-side interface of the system 300includes a tumor procurement module 114 which enables personnel at theclinical facility to obtain the solid tumor from the patient inaccordance with a predetermined protocol, and enter information relatingto the procedure for obtaining the solid tumor from the patient.

In some embodiments, the procurement module 114 includes tumorprocurement forms that are used by the personnel at the clinicalfacility to ensure that appropriate procedure is followed during theprocess of obtaining the solid tumor (or a fragment thereof) from thepatient and preparing it for transportation to the manufacturingfacility. FIGS. 3K-3P are representative screenshots of the tumorprocurement forms according to some embodiments of the presentdisclosure. The tumor procurement forms may, in some embodiments,require a technician obtaining the tumor from the patient to enterinformation relating to the procedure being performed. The enteredinformation may be used for updating batch records for post-facto auditif necessary.

Moreover, because the information included in the batch records isaccessible throughout the system (with appropriate permissions), theinformation relating to the process followed for obtaining the tumor isalso available to the personnel at the manufacturing facility. In someembodiments, the personnel at the manufacturing facility may access theinformation relating to the process for obtaining the tumor as a qualitycontrol measure to ensure that the biological material received at themanufacturing facility is suitable for further processing.

In some embodiments, the tumor procurement forms include smart formfeatures that require that certain criteria are met before thetechnician can proceed to a subsequent step in the process. For example,the technician may be required to input information relating to the cellculture media, such as an expiry date, being used during the process,and if the expiry date is prior to current date, the tumor procurementmodule 114 may alert the technician. Further, the tumor procurementmodule 114 may not allow the technician to enter any further informationrelating to the process, thereby forcing the technician to abort theprocess.

Similarly, the tumor procurement forms may require that the technicianhas performed certain steps or procedures before enabling entry ofinformation relating to a subsequent step or procedure. Thus, theprocurement module 114 requires that the technician follow a certainprotocol without missing or skipping certain steps.

In some embodiments, the procurement module 114 may require that thetumor procurement forms are verified before enabling the release of theobtained tumor to the courier for transportation to the manufacturingfacility. The verification requirement functions as a fail-safe toensure that appropriate protocols and procedures are followed whenobtaining the tumor. In some embodiments, the procurement module 114 mayrequire that the person verifying the tumor procurement forms is not thesame as the person entering the information in the tumor procurementforms. Such verification requirement also ensures compliance withappropriate regulatory requirements.

G. Patient Support Services

Embodiments of the present disclosure are further operable to enablepatient support services that interface with a patient or a patient'srepresentative (collectively referred to herein as the patient) insupport of travel, health management, health insurance, reimbursement,and other treatment related services. The patient support services may,in some embodiments, interface with a telephony interface and a personafrom which the manufacturing process and the COC/COI process isaccessible (with appropriate permissions). As used herein, the termpersona refers to a user type. A given persona is provided with certainlevel of access to the system and the information within the system, andcan access certain functions. The personas within the system include,but are not limited to, Community Persona, Tissue Procurement Persona,Manufacturer Persona, Patient Support Persona, Patient Persona, andHealth System User persona.

The Community persona relates to community users, which is accessible tothe personnel at the center providing the treatment procedures to thepatient. Users of the Community persona may include, for example, celltherapy coordinators, bone marrow transplant nurses, hospital billingdepartment, etc. Functions associated with the users of the Communitypersona include, but are not limited to, registering a new patient,updating patient enrollment data, creating and submitting a TIL orderrequest for a patient, scheduling or changing a resection date, viewingGolden Path dates, view final product delivery dates, uploading hospitalpurchase orders, uploading patient consent forms, enrolling a patientfor patient support options, viewing and updating caregiver records,completing and submitting tumor procurement forms for approval,approving tumor procurement forms, printing tumor procurement forms,uploading tumor procurement forms, and printing media bottle and tumorshipper labels.

Users associated with the Manufacturer persona include tumor receivingpersonnel, quality control personnel, manufacturing process personnel,and final product packaging personnel. Functions associated with theManufacturer persona include, but are not limited to, entering lotnumbers, viewing available and reserved manufacturing slots, printingand scanning in-process, final product and shipping labels.

Users associated with the tumor procurement persona include, forexample, surgeons, and bone marrow transplant nurses. Functionsassociated with the tumor procurement persona may include, withoutlimitation, completing tumor procurement forms, submitting tumorprocurement forms for approval, approving tumor procurement forms,printing tumor procurement forms, and uploading tumor procurement forms.

Users of patient support persona may include, but are not limited to,patient support personnel, patient counsellors, hospital billing,hospital insurance managers, etc. Functions associated with the patientsupport persona may include, but are not limited to, viewing patientassistance services cases, uploading files to the cases, annotating thecases, transferring the cases to a case manager, etc.

In some embodiments of the system, certain types of information isaccessible to certain personas. In some embodiments, the information maybe accessible to users of certain personas only when performing certaintypes of functions, and not when performing other types of functions.For example, the some embodiments, the COC/COI and manufacturing processinformation may be accessible via patient support persona to hospitalbilling and/or hospital insurance managers, but not visible to patientcounsellors. In some embodiments, the manufacturing and COC/COIinformation may be visible to, but not editable by patient supportpersonnel who perform the functions of, e.g., scheduling patienttreatment events, assisting the patient through the scheduling processand/or assisting the patient with transportation for patient treatmentevents.

For example, in some embodiments, a method for manufacturing a celltherapy product by expanding a population of cells obtained from a tumorfrom a patient into the cell therapy product may comprise:

receiving a population of cells from the patient at a manufacturingfacility based on a cell order request to manufacture the cell therapyproduct for the patient;

generating, by a computing device, a patient-specific identifierincluding a cell order identifier associated with the cell orderrequest;

initiating a process to manufacture the cell therapy product, theprocess comprising:

after receiving the population of cells at the manufacturing facility,scheduling, by the computing device, patient treatment events,

initiating expansion of the cell therapy product from at least some ofthe population of cells using a cell expansion technique and determiningacceptance parameters for the expansion cell therapy product at a firsttime point and at a second time point subsequent to the first timepoint,determining whether acceptance parameters for the expansion cell therapyproduct meet acceptance criteria associated with a corresponding timepoint,in response to a determination that the acceptance parameters for theexpansion cell therapy product meet the acceptance criteria at the firsttime point, continuing the expansion of cell therapy product from the atleast some of the obtained cell therapy product using the cell expansiontechnique up to the second time point, andin response to a determination that the acceptance parameters for theexpansion cell therapy product do not meet the acceptance criteria atthe second time point:determining whether re-performing the expansion of the cell therapyproduct using the cell expansion technique is feasible from the firsttime point based on the acceptance parameters at the second time point,in response to a determination that the re-performing is feasible,re-performing the expansion of the cell therapy product from at leastsome of the cell therapy product obtained at the second time point usingthe cell expansion technique from the first time point to obtain thecell therapy product,estimating, by the computing device, a time of completion of theexpansion of the cell therapy product following the re-performing of theexpansion of the cell therapy product from the first time point, andrescheduling, by the computing device, the patient treatment events andcompleting a subsequent expansion of cell therapy product from the firsttime point,wherein the rescheduling of the patient treatment events is performedbased on the estimated time of completion of the expansion of the celltherapy product and a timing of patient treatment events prior to orsubsequent to an infusion of the expanded cell therapy product in thepatient, andproviding patient support services such as, for example, support fortravel, health management, health insurance, reimbursement, and othertreatment related services.

VI. Further Considerations

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the compositions, systems and methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Modifications of the above-described modesfor carrying out the invention that are obvious to persons of skill inthe art are intended to be within the scope of the following claims. Allpatents and publications mentioned in the specification are indicativeof the levels of skill of those skilled in the art to which theinvention pertains.

All headings and section designations are used for clarity and referencepurposes only and are not to be considered limiting in any way. Forexample, those of skill in the art will appreciate the usefulness ofcombining various aspects from different headings and sections asappropriate according to the spirit and scope of the invention describedherein.

All references cited herein are hereby incorporated by reference hereinin their entireties and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this application can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments and examplesdescribed herein are offered by way of example only, and the applicationis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which the claims are entitled.

Various examples of aspects of the disclosure are described as numberedclauses (1, 2, 3, etc.) for convenience. These are provided as examples,and do not limit the subject technology. Identifications of the figuresand reference numbers are provided below merely as examples and forillustrative purposes, and the clauses are not limited by thoseidentifications.

Clause 1. A method for manufacturing a cell therapy product by expandinga population of cells obtained from a tumor from a patient into the celltherapy product, the method comprising:

receiving a population of cells from the patient at a manufacturingfacility based on a cell order request to manufacture the cell therapyproduct for the patient;

generating, by a computing device, a patient-specific identifierincluding a cell order identifier associated with the cell orderrequest;

initiating a process to manufacture the cell therapy product, theprocess comprising: after receiving the population of cells at themanufacturing facility, scheduling, by the computing device, patienttreatment events,

initiating expansion of the cell therapy product from at least some ofthe population of cells using a cell expansion technique and determiningacceptance parameters for the expansion cell therapy product at a firsttime point and at a second time point subsequent to the first timepoint,determining whether acceptance parameters for the expansion cell therapyproduct meet acceptance criteria associated with a corresponding timepoint,in response to a determination that the acceptance parameters for theexpansion cell therapy product meet the acceptance criteria at the firsttime point, continuing the expansion of cell therapy product from the atleast some of the obtained cell therapy product using the cell expansiontechnique up to the second time point, andin response to a determination that the acceptance parameters for theexpansion cell therapy product do not meet the acceptance criteria atthe second time point:determining whether re-performing the expansion of the cell therapyproduct using the cell expansion technique is feasible from the firsttime point based on the acceptance parameters at the second time point,in response to a determination that the re-performing is feasible,re-performing the expansion of the cell therapy product from at leastsome of the cell therapy product obtained at the second time point usingthe cell expansion technique from the first time point to obtain thecell therapy product,estimating, by the computing device, a time of completion of theexpansion of the cell therapy product following the re-performing of theexpansion of the cell therapy product from the first time point, andrescheduling, by the computing device, the patient treatment events andcompleting a subsequent expansion of cell therapy product from the firsttime point,wherein the rescheduling of the patient treatment events is performedbased on the estimated time of completion of the expansion of the celltherapy product and a timing of patient treatment events prior to orsubsequent to an infusion of the expanded cell therapy product in thepatient.

Clause 2. The method of clause 1, wherein the cell therapy productcomprises T-cells.

Clause 3. The method of clause 1, wherein the cell therapy productcomprises tumor infiltrating lymphocytes (TILs).

Clause 4. The method of clause 1, wherein the patient treatment eventsinclude one or more of an inpatient stay time period, resection date,lymphodepletion date, infusion date for infusing the patient with thecell therapy product and IL-2 treatment date.

Clause 5. The method of clause 1, wherein the determining whether theacceptance parameters for the expansion cell therapy product meet theacceptance criteria comprises determining the acceptance parameters forthe expansion cell therapy product at a plurality of time pointsfollowing the initiation of the expansion of the obtained cell therapyproduct, the plurality of time points including the first and the secondtime points.

Clause 6. The method of clause 5, wherein the rescheduling of patienttreatment events comprises rescheduling the patient treatment events inresponse to a determination that the acceptance parameters for theexpansion cell therapy product do not meet the acceptance criteria atany of the plurality of time points.

Clause 7. The method of clause 5, wherein the rescheduling of patienttreatment events comprises terminating the patient treatment events inresponse to a determination that the acceptance parameters for theexpansion cell therapy product do not meet the acceptance criteria atany of the plurality of time points because of contamination.

Clause 8. The method of clause 5, wherein in response to a determinationthat the acceptance parameters for the expansion cell therapy product donot meet the acceptance criteria at any of the plurality of time pointsbecause of contamination, terminating the subsequent expansion of celltherapy product.

Clause 9. The method of clause 8, wherein determining acceptanceparameters for the expansion cell therapy product comprise one or moreof determination of viability, sterility, cell count, mycoplasma count,CD3 count, result of an Endotoxin assay, and a result of a Gram stainassay.

Clause 10. The method of clause 9, wherein the cell expansion techniquecomprises a rapid expansion step, and

the method further comprises:

determining whether the acceptance parameters for the expansion celltherapy product meet the acceptance criteria prior to the rapidexpansion step; and

in response to a determination that the acceptance parameters for theexpansion cell therapy product meet the acceptance criteria, schedulinga lymphodepletion date at a date prior to the completion of themanufacturing of the expanded cell therapy product, scheduling aninfusion date at a date following the completion of the manufacturing ofthe expanded cell therapy product, and scheduling an IL-2 treatment datefollowing the infusion date.

Clause 11. The method of clause 1, wherein the cell expansion techniqueincludes culturing the cell therapy product in a single closed containerbioreactor.

Clause 12. The method of clause 1, wherein estimating the time ofcompletion of the expansion of the cell therapy product following there-performing of the expansion of the cell therapy product from thefirst time point comprises determining the time needed for completingthe expansion process from the first time point based on one or both ofthe acceptance parameters at the second time point and the acceptanceparameters associated with a population of cells used for re-performingthe expansion of the cell therapy product from the first time point.

Clause 13. The method of clause 1, wherein cell order request to expandcell therapy product is received from a hospital-side interface, and themethod further comprises transmitting, upon receiving the cell orderrequest, a confirmation, including one or both of the patient-specificidentifier and the cell order identifier, to the hospital-side interfacethat the cell order request associated with the patient has beenreceived.

Clause 14. The method of clause 13, further comprising scheduling a setof dates corresponding to a plurality of time points, including thefirst and second time points, for determining whether acceptanceparameters for the expansion cell therapy product meet the acceptancecriteria during expansion of the cell therapy product depending on thecell expansion technique and when the cell order request is received.

Clause 15. The method of clause 13, further comprising transmitting,upon rescheduling the patient treatment events, to the hospital-sideinterface an updated schedule for the patient treatment eventsassociated with the patient-specific identifier.

Clause 16. The method of clause 15, further comprising:

transmitting, to a logistics interface, a pick-up order associated withthe patient-specific identifier based on the time of completion; and

transmitting, to a hospital-side interface, a schedule for the patienttreatment events associated with the patient-specific identifier basedon the time of completion.

Clause 17. The method of clause 16, further comprising: in response to adetermination that re-performing of the expansion of the cell therapyproduct from the first time point is feasible:

disassociating, by the computing device, the cell order identifier fromthe patient-specific identifier, and

generating, by the computing device, a new cell order identifierassociated with the cell order request and associating the new cellorder identifier with the patient-specific identifier.

Clause 18. The method of clause 17, wherein the cell order request toexpand cell therapy product is received from a hospital-side interface,and the method further comprises transmitting the patient-specificidentifier including the new cell order identifier and the estimatedtime of completion of the expansion of the cell therapy product to thehospital-side interface.

Clause 19. The method of clause 17, further comprising:

generating, by the computing device, a new schedule for shipping andlogistics events associated with the patient treatment events based onthe rescheduling of the patient treatment events, and

transmitting the new schedule of shipping and logistics eventsassociated the patient-specific identifier including the new cell orderidentifier to a logistics interface based on the rescheduling ofshipping and logistics events.

Clause 20. The method of clause 17, further comprising:

associating, by the computing device, with the patient-specificidentifier at each time point at which the determination of whetheracceptance parameters meet the certain acceptance criteria is made, thenew cell order identifier including fields corresponding to eachrespective time point and a result of the determination.

Clause 21. The method of clause 20, wherein the scheduling comprises:

scheduling, by the computing device, the patient treatment events basedon the patient-specific identifier including the new cell orderidentifier.

Clause 22. The method of clause 21, further comprising, in response to adetermination that the re-performing is not possible, canceling, by thecomputing device, the patient treatment events scheduled subsequent tothe second time point.

Clause 23. The method of clause 22, further comprising transmitting thecancellation of patient treatment events to a hospital-side interfaceand a logistics interface.

Clause 24. The method of clause 23, further comprising, in response to adetermination that the re-performing is not feasible, destroying theexpanded cell therapy product and disassociating the patient-specificidentifier from the cell order identifier.

Clause 25. A method of treating the patient with the expansion celltherapy product obtained by the method of clause 1 in accordance withthe rescheduled patient treatment events.

Clause 26. A method of manufacturing a cell therapy product for apatient, the method comprising:

receiving, at a computing device, a cell order request to manufacturethe cell therapy product for the patient, manufacturing slots at aplurality of manufacturing facilities for manufacturing the cell therapyproduct, wherein the manufacturing slots for a respective manufacturingfacility are received at the computing device from a manufacturercomputer subsystem associated with the respective manufacturingfacility, and a preliminary schedule of patient treatment events fortreating the patient with the cell therapy product;determining and displaying in a scheduling user interface, by thecomputing device, a plurality of available manufacturing slots formanufacturing the cell therapy product based on the preliminary scheduleof patient treatment events;after selection of one of the available manufacturing slots, performing,at a medical facility, a procedure on the patient to obtain a solidtumor from the patient in accordance with the preliminary schedule ofpatient treatment events and the available manufacturing slot;transferring the obtained solid tumor to a manufacturing facilitycorresponding to the available manufacturing slot in accordance with theavailable manufacturing slot;initiating, upon receiving the obtained solid tumor at the manufacturingfacility, manufacturing of the cell therapy product from at least aportion of the obtained solid tumor using a cell expansion technique;determining, during the manufacturing of the cell therapy product,acceptance parameters for the manufactured cell therapy product at afirst time point and a second time point subsequent to the first timepoint and whether the acceptance parameters meet acceptance criteriaassociated with a corresponding time point, the acceptance parametersbeing determined based on a result of an assay associated with thecorresponding time point;modifying, by the computing device, a manufacturing schedule formanufacturing of the cell therapy product, manufacturing slotscorresponding to the manufacturing facility, and the preliminaryschedule of patient treatment events based on whether the acceptancecriteria at one or both of the first and second time points are met; andcompleting the manufacturing of the cell therapy product in accordancewith the modified manufacturing schedule if the acceptance criteria atboth the first and second time points are met.

Clause 27. The method of clause 26, further comprising transferring themanufactured cell therapy product to the medical facility.

Clause 28. The method of clause 26, further comprising, upon receivingthe cell order request, generating, by the computing device, apatient-specific identifier associated with the patient and the cellorder request.

Clause 29. The method of clause 28, further comprising automaticallygenerating, by the computing device, a shipping label for a containerfor the obtained solid tumor, the shipping label comprising thepatient-specific identifier, the cell order request, the manufacturingslot and the manufacturing facility corresponding to the availablemanufacturing slot.

Clause 30. The method of clause 29, further comprising automaticallygenerating, by the computing device, a manufacturing label for thecontainer for the obtained solid tumor, the manufacturing labelcomprising a time point corresponding to a manufacturing process beingused for manufacturing the cell therapy product when the manufacturinglabel is generated, a container-identifying bar code, thepatient-specific identifier, manufacturing steps completed at the timepoint, acceptance parameters associated with the completed processes,and a manufacturing process being performed at the time point.

Clause 31. The method of clause 26, wherein the cell therapy productcomprises T-cells.

Clause 32. The method of clause 26, wherein the cell therapy productcomprises tumor infiltrating lymphocytes (TILs).

Clause 33. The method of clause 26, wherein the patient treatment eventsinclude one or more of an inpatient stay time period, resection date,lymphodepletion date, infusion date for infusing the patient with thecell therapy product and IL-2 treatment date.

Clause 34. The method of clause 26, wherein the determining whether theacceptance parameters for the manufactured cell therapy product meet theacceptance criteria comprises determining the acceptance parameters forthe manufactured cell therapy product at a plurality of time pointsfollowing the initiation of the expansion of the received cell therapyproduct, the plurality of time points including the first and the secondtime points.

Clause 35. The method of clause 34, further comprising: rescheduling, bythe computing device, the patient treatment events and completing asubsequent expansion of cell therapy product, wherein the reschedulingof patient treatment events comprises rescheduling the patient treatmentevents in response to a determination that the acceptance parameters forthe manufactured cell therapy product do not meet the acceptancecriteria at any of the plurality of time points.

Clause 36. The method of clause 34, wherein the rescheduling of patienttreatment events comprises terminating the patient treatment events inresponse to a determination that the acceptance parameters for themanufactured cell therapy product do not meet the acceptance criteria atany of the plurality of time points because of contamination.

Clause 37. The method of clause 34, wherein in response to adetermination that the acceptance parameters for the manufactured celltherapy product do not meet the acceptance criteria at any of theplurality of time points because of contamination, terminating thesubsequent expansion of cell therapy product.

Clause 38. The method of clause 37, wherein determining acceptanceparameters for the manufactured cell therapy product comprise one ormore of determination of viability, sterility, cell count, mycoplasmacount, CD3 count, result of an Endotoxin assay, and a result of a Gramstain assay.

Clause 39. The method of clause 38, wherein the cell expansion techniquecomprises a rapid expansion step, and

the method further comprises:

determining whether the acceptance parameters for the manufactured celltherapy product meet the acceptance criteria prior to the rapidexpansion step; and

in response to a determination that the acceptance parameters for themanufactured cell therapy product meet the acceptance criteria,scheduling a lymphodepletion date at a date prior to the completion ofthe manufacturing of the cell therapy product, scheduling an infusiondate at a date following the completion of the manufacturing of the celltherapy product, and scheduling an IL-2 treatment date following theinfusion date.

Clause 40. The method of clause 26, wherein the cell expansion techniqueincludes culturing the cell therapy product in a single closed containerbioreactor.

Clause 41. The method of clause 26, further comprising: estimating, bythe computing device, a time of completion of the expansion of the celltherapy product, wherein estimating the time of completion of theexpansion of the cell therapy product following a re-performing of theexpansion of the cell therapy product from the first time pointcomprises determining the time needed for completing the expansionprocess from the first time point based on one or both of the acceptanceparameters at the second time point and the acceptance parametersassociated with a population of cells used for re-performing theexpansion of the cell therapy product from the first time point.

Clause 42. The method of clause 29, wherein cell order request to expandcell therapy product is received from a hospital-side interface, and themethod further comprises transmitting, upon receiving the cell orderrequest, a confirmation, including one or both of the patient-specificidentifier and the cell order identifier, to the hospital-side interfacethat the cell order request associated with the patient has beenreceived.

Clause 43. The method of clause 42, further comprising scheduling a setof dates corresponding to a plurality of time points, including thefirst and second time points, for determining whether acceptanceparameters for the manufactured cell therapy product meet the acceptancecriteria during expansion of the cell therapy product depending on thecell expansion technique and when the cell order request is received.

Clause 44. The method of clause 43, further comprising transmitting,upon rescheduling the patient treatment events, to the hospital-sideinterface an updated schedule for the patient treatment eventsassociated with the patient-specific identifier.

Clause 45. The method of clause 44, further comprising:

transmitting, to a logistics interface, a pick-up order associated withthe patient-specific identifier based on the time of completion; and

transmitting, to a hospital-side interface, a schedule for the patienttreatment events associated with the patient-specific identifier basedon the time of completion.

Clause 46. The method of clause 45, further comprising: in response to adetermination that re-performing of the expansion of the cell therapyproduct from the first time point is feasible:

disassociating, by the computing device, the cell order identifier fromthe patient-specific identifier, and

generating, by the computing device, a new cell order identifierassociated with the cell order request and associating the new cellorder identifier with the patient-specific identifier.

Clause 47. The method of clause 46, wherein the cell order request toexpand cell therapy product is received from a hospital-side interface,and the method further comprises transmitting the patient-specificidentifier including the new cell order identifier and an estimated timeof completion of the expansion of the cell therapy product to thehospital-side interface.

Clause 48. The method of clause 47, further comprising:

generating, by the computing device, a new schedule for shipping andlogistics events associated with the patient treatment events based onthe rescheduling of the patient treatment events, and

transmitting the new schedule of shipping and logistics eventsassociated the patient-specific identifier including the new cell orderidentifier to a logistics interface based on the rescheduling ofshipping and logistics events.

Clause 49. The method of clause 48, further comprising:

associating, by the computing device, with the patient-specificidentifier at each time point at which the determination of whetheracceptance parameters meet the acceptance criteria is made, the new cellorder identifier including fields corresponding to each respective timepoint and a result of the determination.

Clause 50. The method of clause 49, wherein the scheduling comprises:

scheduling, by the computing device, the patient treatment events basedon the patient-specific identifier including the new cell orderidentifier.

Clause 51. The method of clause 50, further comprising, in response to adetermination that the re-performing is not possible, canceling, by thecomputing device, the patient treatment events scheduled subsequent tothe second time point.

Clause 52. The method of clause 51, further comprising transmitting thecancellation of patient treatment events to a hospital-side interfaceand a logistics interface.

Clause 53. The method of clause 52, further comprising, in response to adetermination that the re-performing is not feasible, destroying theexpanded cell therapy product and disassociating the patient-specificidentifier from the cell order identifier.

Clause 54. A method of treating the patient with the manufactured celltherapy product obtained by the method of clause 26 in accordance withthe rescheduled patient treatment events.

Clause 55. A method for manufacturing a cell therapy product, the methodcomprising:

receiving, at a manufacturing facility, a solid tumor obtained from apatient;

generating, by a computing device, a manufacturing label for amanufacturing container to be used in a process for manufacturing thecell therapy product from at least a portion of the obtained solid tumorusing a cell expansion technique, the manufacturing label comprisinginformation associated with the patient, the manufacturing process andquality of manufactured cell therapy product;initiating a process to manufacture the cell therapy product, theprocess comprising:performing, at a medical facility, a procedure on the patient to obtaina solid tumor from the patient,transferring the obtained solid tumor to a manufacturing facility, afterreceiving the obtained solid tumor at the manufacturing facility,dynamic scheduling, by the computing device, patient treatment events,the dynamic scheduling being dependent on acceptance parameters forsubsequently obtained expansion cell therapy product,initiating expansion of the cell therapy product from at least some ofthe obtained solid tumor using a cell expansion technique anddetermining acceptance parameters for the expansion cell therapy productat a plurality of time points,performing a quality control assay to determine acceptance parametersfor the manufactured cell therapy product at the plurality of timepoints;receiving, at the computing device, the acceptance parameters for themanufactured cell therapy product;generating, by the computing device, an updated manufacturing labelcorresponding to each of the plurality of time points, the updatedmanufacturing label comprising updated information associated withquality of manufactured cell therapy product, the updated informationcomprising the acceptance parameters at a corresponding time point;reading, by the computing device, the updated manufacturing label ateach of the plurality of time points; andcompleting expansion of the cell therapy product based on informationread from the updated manufacturing label at each of the plurality oftime points.

Clause 56. The method of clause 55, further comprising providing, by thecomputing device, a warning signal if:

information relating to the patient on the updated manufacturing labelfor a subsequent manufacturing step does not match the informationrelating to the patient on the manufacturing label for an immediatelypreceding manufacturing step, or

acceptance parameters on the updated manufacturing label for a giventime point in the manufacturing process do not meet acceptance criteriafor that time point in the manufacturing process,

wherein the acceptance parameters comprise one or more of viability,sterility, cell count, mycoplasma count, CD3 count, a result of anendotoxin assay, and a result of a Gram stain assay.

Clause 57. The method of clause 55, wherein information relating to thepatient comprises a patient-specific identifier and a cell orderidentifier associated with a cell order request to manufacture the celltherapy product for the patient.

Clause 58. The method of clause 55, wherein the cell expansion techniqueincludes culturing the cell therapy product in a single closed containerbioreactor.

Clause 59. The method of clause 55, wherein the manufacturing labelcomprises a barcode encoding the information associated with thepatient, the manufacturing process and quality of manufactured celltherapy product.

Clause 60. The method of clause 56, further comprising scheduling a setof dates corresponding to a plurality of time points, including a firsttime point and a second time point subsequent to the first time point,for determining whether acceptance parameters for the manufactured celltherapy product meet the acceptance criteria during the manufacturingprocess depending on the cell expansion technique being used and when acell order request is received at the manufacturing facility.

Clause 61. The method of clause 60, further comprising integrating witha logistics interface, receiving courier status information via acourier computing subsystem, wherein courier status information includesand in response to receiving the courier status information, determiningshipping schedule for shipping the manufactured cell therapy productbased on the determined schedule of manufacturing and generating ashipping label for a shipping container containing the manufactured celltherapy product.

Clause 62. The method of clause 60, further comprising transmitting ashipping request to a logistics facility based on the determinedshipping schedule.

Clause 63. The method of clause 60, further comprising generating ashipping label for a shipping container containing the manufactured celltherapy product before performing a final quality control assay, theshipping label being indicative that the manufactured cell therapyproduct is not releasable unless a result of the final quality controlassay indicates that the corresponding acceptance parameters meet thecorresponding acceptance criteria.

Clause 64. The method of clause 56, further comprising:

upon determining that the acceptance parameters for the manufacturedcell therapy product meet the acceptance criteria, determining acompletion date for the manufacturing of the cell therapy;

generating, by the computing device, a schedule for patient treatmentevents corresponding to a use of the cell therapy product for treating apatient based on the completion date;

transmitting, to a logistics interface, a pick-up order based on thecompletion date; and

transmitting, to a hospital-side interface, the schedule for the patienttreatment events.

Clause 65. The method of clause 55, wherein the cell therapy productcomprises T-cells.

Clause 66. The method of clause 55, wherein the cell therapy productcomprises tumor infiltrating lymphocytes (TILs).

Clause 67. The method of clause 55, wherein the patient treatment eventsinclude one or more of an inpatient stay time period, resection date,lymphodepletion date, infusion date for infusing the patient with thecell therapy product and IL-2 treatment date.

Clause 68. The method of clause 56 wherein the determining whether theacceptance parameters for the expansion cell therapy product meet theacceptance criteria comprises determining the acceptance parameters forthe expansion cell therapy product at a plurality of time pointsfollowing the initiation of the expansion of the obtained cell therapyproduct, the plurality of time points including the first and the secondtime points.

Clause 69. The method of clause 68, further comprising: rescheduling, bythe computing device, the patient treatment events and completing asubsequent expansion of cell therapy product, wherein the reschedulingof patient treatment events comprises rescheduling the patient treatmentevents in response to a determination that the acceptance parameters forthe expansion cell therapy product do not meet the acceptance criteriaat any of the plurality of time points.

Clause 70. The method of clause 68, wherein the rescheduling of patienttreatment events comprises terminating the patient treatment events inresponse to a determination that the acceptance parameters for theexpansion cell therapy product do not meet the acceptance criteria atany of the plurality of time points because of contamination.

Clause 71. The method of clause 68, wherein in response to adetermination that the acceptance parameters for the expansion celltherapy product do not meet the acceptance criteria at any of theplurality of time points because of contamination, terminating thesubsequent expansion of cell therapy product.

Clause 72. The method of clause 71, wherein determining acceptanceparameters for the expansion cell therapy product comprise one or moreof determination of viability, sterility, cell count, mycoplasma count,CD3 count, result of an Endotoxin assay, and a result of a Gram stainassay.

Clause 73. The method of clause 72, wherein the cell expansion techniquecomprises a rapid expansion step, and

the method further comprises:

determining whether the acceptance parameters for the expansion celltherapy product meet the acceptance criteria prior to the rapidexpansion step; and

in response to a determination that the acceptance parameters for theexpansion cell therapy product meet the acceptance criteria, schedulinga lymphodepletion date at a date prior to the completion of themanufacturing of the cell therapy product, scheduling an infusion dateat a date following the completion of the manufacturing of the celltherapy product, and scheduling an IL-2 treatment date following theinfusion date.

Clause 74. The method of clause 55, wherein the cell expansion techniqueincludes culturing the cell therapy product in a single closed containerbioreactor.

Clause 75. The method of clause 55, wherein estimating the time ofcompletion of the expansion of the cell therapy product following are-performing of the expansion of the cell therapy product from thefirst time point comprises determining the time needed for completingthe expansion process from the first time point based on one or both ofthe acceptance parameters at the second time point and the acceptanceparameters associated with a population of cells used for re-performingthe expansion of the cell therapy product from the first time point.

Clause 76. The method of clause 57, wherein cell order request to expandcell therapy product is received from a hospital-side interface, and themethod further comprises transmitting, upon receiving the cell orderrequest, a confirmation, including one or both of the patient-specificidentifier and the cell order identifier, to the hospital-side interfacethat the cell order request associated with the patient has beenreceived.

Clause 77. The method of clause 76, further comprising scheduling a setof dates corresponding to a plurality of time points, including thefirst and second time points, for determining whether acceptanceparameters for the expansion cell therapy product meet the acceptancecriteria during expansion of the cell therapy product depending on thecell expansion technique and when the cell order request is received.

Clause 78. The method of clause 76, further comprising transmitting,upon rescheduling the patient treatment events, to the hospital-sideinterface an updated schedule for the patient treatment eventsassociated with the patient-specific identifier.

Clause 79. The method of clause 78, further comprising: transmitting, toa logistics interface, a pick-up order associated with thepatient-specific identifier based on the time of completion; andtransmitting, to a hospital-side interface, a schedule for the patienttreatment events associated with the patient-specific identifier basedon the time of completion.

Clause 80. The method of clause 79, further comprising: in response to adetermination that re-performing of the expansion of the cell therapyproduct from the first time point is feasible:

disassociating, by the computing device, the cell order identifier fromthe patient-specific identifier, and

generating, by the computing device, a new cell order identifierassociated with the cell order request and associating the new cellorder identifier with the patient-specific identifier.

Clause 81. The method of clause 80, wherein the cell order request toexpand cell therapy product is received from a hospital-side interface,and the method further comprises transmitting the patient-specificidentifier including the new cell order identifier and an estimated timeof completion of the expansion of the cell therapy product to thehospital-side interface.

Clause 82. The method of clause 80, further comprising:

generating, by the computing device, a new schedule for shipping andlogistics events associated with the patient treatment events based onthe rescheduling of the patient treatment events, and

transmitting the new schedule of shipping and logistics eventsassociated the patient-specific identifier including the new cell orderidentifier to a logistics interface based on the rescheduling ofshipping and logistics events.

Clause 83. The method of clause 80, further comprising:

associating, by the computing device, with the patient-specificidentifier at each time point at which the determination of whetheracceptance parameters meet the certain acceptance criteria is made, thenew cell order identifier including fields corresponding to eachrespective time point and a result of the determination.

Clause 84. The method of clause 83, wherein the scheduling comprises:

scheduling, by the computing device, the patient treatment events basedon the patient-specific identifier including the new cell orderidentifier.

Clause 85. The method of clause 84, further comprising, in response to adetermination that the re-performing is not possible, canceling, by thecomputing device, the patient treatment events scheduled subsequent tothe second time point.

Clause 86. The method of clause 85, further comprising transmitting thecancellation of patient treatment events to a hospital-side interfaceand a logistics interface.

Clause 87. The method of clause 86, further comprising, in response to adetermination that the re-performing is not feasible, destroying theexpanded cell therapy product and disassociating the patient-specificidentifier from the cell order identifier.

Clause 88. A method of treating the patient with the expansion celltherapy product obtained by the method of clause 55 in accordance withthe rescheduled patient treatment events.

What is claimed is:
 1. A method for manufacturing a cell therapy productcomprising tumor infiltrating lymphocytes (TILs) to be used in acomposition for treating a patient having cancer, the method comprising:receiving, at a computing device, a request for manufacturing the celltherapy product, from at least a portion of a solid tumor obtained fromthe patient or a first population of cells obtained from the solidtumor, using a selected multi-step cell expansion process, the requestincluding acceptance criteria for manufactured cell therapy product ateach of a plurality of steps during the selected multi-step cellexpansion process; generating, by the computing device, a containerlabel based on the request, the container label comprising informationassociated with: (a) a manufacturing order received from a treatmentfacility requesting the cell therapy product, including anorder-specific identifier, (b) the patient, including a patient-specificidentifier, (c) the treatment facility requesting the manufacturing ofthe cell therapy product, (d) the selected multi-step cell expansionprocess; and (e) a manufacturing facility where the cell therapy productis to be manufactured; determining, by the computing device, a schedulefor manufacturing the cell therapy product at the manufacturing facilitybased on (i) a time of receipt of the solid tumor or the firstpopulation of cells and (ii) the selected multi-step cell expansionprocess; generating, by the computing device, a manufacturing label tobe affixed to a manufacturing container to be used during the selectedmulti-step cell expansion process, the manufacturing label comprisinginformation associated with: (a) the manufacturing order, including theorder-specific identifier, (b) the patient, including thepatient-specific identifier, (c) the selected multi-step cell expansionprocess or a step thereof and (f) quality of manufactured cell therapyproduct; initiating the selected multi-step cell expansion process tomanufacture the cell therapy product in accordance with the schedule;performing quality control assays to determine acceptance parameters ateach of a plurality of time points during the selected multi-step cellexpansion process; at each of the plurality of time points generating,by the computing device, a respective updated manufacturing label, to beaffixed to the manufacturing container, corresponding to a respectiveone of the plurality of time points, the updated manufacturing labelcomprising information associated with (a) the manufacturing order,including the order-specific identifier, (b) the patient, including thepatient identifier, (c) the selected multi-step cell expansion processand (ee) updated information associated with quality of manufacturedcell therapy product comprising the determined acceptance parameters ata corresponding time point; determining an adjusted schedule, by thecomputing device, for manufacturing the cell therapy product based onwhether the acceptance parameters at a given time point read from theupdated manufacturing label meet the acceptance criteria at the giventime point; and completing the selected multi-step cell expansionprocess in accordance with the adjusted schedule to manufacture the celltherapy product.
 2. The method of claim 1, further comprising, for eachstep of the selected multi-step cell expansion process, providing, bythe computing device, a warning signal when: information relating to thepatient on the updated manufacturing label for a given step does notmatch the information relating to the patient on the manufacturing labelfor a preceding step, or acceptance parameters on the updatedmanufacturing label for a given time point in the selected multi-stepcell expansion process do not meet acceptance criteria for the giventime point in the selected multi-step cell expansion process, whereinthe acceptance parameters comprise one or more of viability, sterility,cell count, mycoplasma count, CD3 count, a result of an endotoxin assay,and a result of a Gram stain assay.
 3. The method of claim 1, whereinthe selected multi-step cell expansion process includes culturing theTILs obtained from the solid tumor or the first population of cells in aclosed container bioreactor.
 4. The method of claim 1, wherein themanufacturing label comprises a barcode encoding the informationassociated with the patient, the selected multi-step cell expansionprocess and quality of manufactured cell therapy product.
 5. The methodof claim 1, wherein determining the schedule for manufacturing the celltherapy product comprises scheduling a set of dates corresponding to aplurality of time points, including a first time point and a second timepoint subsequent to the first time point, for determining whetheracceptance parameters for the manufactured cell therapy product meet theacceptance criteria during the selected multi-step cell expansionprocess to manufacture the cell therapy product depending on theselected multi-step cell expansion technique being used process and whenthe manufacturing order is received at the manufacturing facility. 6.The method of claim 1, further comprising receiving courier statusinformation via the computing device; in response to receiving thecourier status information, determining shipping schedule for shippingthe manufactured cell therapy product based on the determined adjustedschedule of manufacturing, and generating a shipping label for ashipping container containing the manufactured cell therapy product. 7.The method of claim 6, further comprising transmitting, by the computingdevice, a shipping request to a logistics facility based on thedetermined shipping schedule.
 8. The method of claim 1, furthercomprising: upon determining that the acceptance parameters for themanufactured cell therapy product meet the acceptance criteria, by thecomputing device, determining a completion date for the manufacturing ofthe cell therapy product; generating, by the computing device, aschedule for patient treatment events corresponding to a use of the celltherapy product for treating a patient based on the completion date;transmitting, by the computing device, to a logistics interface, apick-up order based on the completion date; and transmitting, by thecomputing device, to a hospital-side interface, the schedule for thepatient treatment events.
 9. The method of claim 1, wherein themanufacturing label further comprises a given time point correspondingto a given step of the selected multi-step cell expansion process beingwhen the manufacturing label is generated, a container-identifying code,steps completed at the given time point, acceptance parametersassociated with the completed steps, and a step to be performed at orafter the given time point.
 10. The method of claim 1, wherein themanufacturing label comprises a graphical identification code encoding(a) a manufacturing order received from a treatment facility requestingthe cell therapy product, (b) the patient, including a patientidentifier, (c) the selected multi-step cell expansion process and (d)quality of manufactured cell therapy product, the graphicalidentification code being one of a 1-dimensional barcode and a2-dimensional barcode.
 11. The method of claim 1, further comprisinggenerating a contingent shipping label for a shipping containercontaining the cell therapy product before performing a final qualitycontrol assay, the contingent shipping label being indicative that thecell therapy product is not releasable unless a result of the finalquality control assay indicates that the acceptance parameters meetacceptance criteria.
 12. A method for manufacturing a cell therapyproduct comprising tumor infiltrating lymphocytes (TILs) for treatmentof cancer, the method comprising: receiving, at a computing device, anindication that a solid tumor obtained from a patient having the canceror a first population of cells comprising TILs obtained from the solidtumor obtained from the patient has been received at a manufacturingfacility for manufacturing the cell therapy product using a selectedmulti-step cell expansion process; generating, by the computing device,a manufacturing label to be affixed to a manufacturing container to beused during a multi-step cell expansion process for manufacturing thecell therapy product from at least a portion of the obtained solid tumoror the first population of cells, the manufacturing label comprisinginformation associated with: (a) a manufacturing order received from atreatment facility requesting the cell therapy product, including anorder-specific identifier, (b) the patient, including a patient-specificidentifier, (c) the selected multi-step cell expansion process and (d)quality of manufactured cell therapy product; initiating the multi-stepcell expansion process to manufacture the cell therapy product;performing a quality control assay to determine acceptance parametersfor the manufactured cell therapy product at each of a plurality of timepoints during the selected multi-step cell expansion process; receiving,at the computing device, the acceptance parameters for the manufacturedcell therapy product at each of the plurality of time points; reading,by the computing device, the manufacturing label at each of theplurality of time points; and for each step of the multi-step cellexpansion process, generating, by the computing device, a warning signalwhen: information relating to the patient on the manufacturing label forthe step does not match the information relating to the patient on themanufacturing label for an immediately preceding manufacturing step, oracceptance parameters on the manufacturing label for a given time pointin the selected multi-step cell expansion process do not meet acceptancecriteria for the given time point in the selected multi-step cellexpansion process.
 13. The method of claim 12, wherein the acceptanceparameters comprise one or more of viability, sterility, cell count,mycoplasma count, CD3 count, a result of an endotoxin assay, and aresult of a Gram stain assay.
 14. The method of claim 12, furthercomprising generating, by the computing device, an updated manufacturinglabel corresponding to each of the plurality of time points, the updatedmanufacturing label further comprising updated information associatedwith quality of manufactured cell therapy product, the updatedinformation comprising the acceptance parameters at a corresponding timepoint.
 15. The method of claim 12, wherein the warning signal comprises:when information relating to the patient on the manufacturing label forthe manufacturing step does not match the information relating to thepatient on the manufacturing label for the immediately precedingmanufacturing step, a prevention signal preventing performance ofsubsequent steps in the multi-step cell expansion process.
 16. Themethod of claim 12, wherein the warning signal comprises: if acceptanceparameters on the manufacturing label for a given time point in theselected multi-step cell expansion process do not meet acceptancecriteria for that time point in the selected multi-step cell expansionprocess, an alert signal indicating that the acceptance criteria for thegiven time point are not met.
 17. The method of claim 16, wherein thealert signal causes the computing device to generate an updatedmanufacturing label comprising updated information associated withquality of manufactured cell therapy product, the updated informationcomprising the acceptance parameters at the given time point.
 18. Themethod of claim 17, further comprising reading the updated manufacturinglabel and determining, based on the updated information, whetheradditional processing of the manufactured cell therapy product can beperformed to meet the acceptance criteria at that time point.
 19. Themethod of claim 18, further comprising controlling, by the computingdevice, the multi-step cell expansion process to enable the additionalprocessing of the manufactured cell therapy product to meet theacceptance criteria at the given time point.